Thyroid Hormone Analogs and Methods of Use

ABSTRACT

Disclosed are methods of treating subjects having conditions related to angiogenesis including administering an effective amount of a polymeric form of thyroid hormone, or an antagonist thereof, to promote or inhibit angiogenesis in the subject. Compositions of the polymeric forms of thyroid hormone, or thyroid hormone analogs, are also disclosed.

FIELD OF THE INVENTION

This invention relates to thyroid hormone, thyroid hormone analogs andderivatives, and polymeric forms thereof. Methods of using suchcompounds, and pharmaceutical compositions containing same are alsodisclosed. The invention also relates to methods of preparing suchcompounds.

BACKGROUND OF THE INVENTION

Thyroid hormones, L-thyroxine (T4) and L-triiodothyronine (T3), regulatemany different physiological processes in different tissues invertebrates. Most of the actions of thyroid hormones are mediated by thethyroid hormone receptor (“TR”), which is a member of the nuclearreceptor superfamily of ligand-activated transcription regulators. Thissuperfamily also includes receptors for steroid hormones, retinoids, and1,25-dihydroxy vitamin D3. These receptors are transcription factorsthat can regulate expression of specific genes in various tissues andare targets for widely used drugs, such as tamoxifen, an estrogenreceptor partial antagonist. There are two different genes that encodetwo different TRs, TRα and TRβ. These two TRs are often co-expressed atdifferent levels in different tissues. Most thyroid hormones do notdiscriminate between the two TRs and bind both with similar affinities.

Gene knockout studies in mice indicate that TRβ plays a role in thedevelopment of the auditory system and in the negative feedback ofthyroid stimulating hormone by T3 in the pituitary, whereas TRα,modulates the effect of thyroid hormone on calorigenesis and on thecardiovascular system. The identification of TR antagonists could playan important role in the future treatment of hypothyroidism. Suchmolecules would act rapidly by directly antagonizing the effect ofthyroid hormone at the receptor level, a significant improvement forindividuals with hypothyroidism who require surgery, have cardiacdisease, or are at risk for life-threatening thyrotoxic storm.

Thus, there remains a need for the development of compounds thatselectively modulate thyroid hormone action by functioning asisoform-selective agonists or antagonists of the thyroid hormonereceptors (TRs) would prove useful for medical therapy. Recent effortshave focused on the design and synthesis of thyroid hormone (T3/T4)antagonists as potential therapeutic agents and chemical probes. Thereis also a need for the development of thyromimetic compounds that aremore accessible than the natural hormone and have potentially usefulreceptor binding and activation properties.

Thyroid hormone receptor preferentially binds 3,5,3′-triiodo-L-thyronine(T3), a hormone analogue derived by tissue deiodination of circulatingL-thyroxine (T4). However, the ability of T4 and T3 to activateintracellular signal transduction cascades, independently of TR, hasrecently been described by several laboratories. Acting independently ofTR, thyroid hormone also modulates activity of the plasma membraneNa⁺/H⁺ exchanger, Ca²⁺-stimulable ATPase, several other ion pumps orchannels, and GTPase activity of synaptosomes. Studies from severallaboratories have demonstrated the ability of thyroid hormone toactivate the MAPK signal transduction cascade. These pathways typicallyare activated by physical and chemical signals at the cell surface.Although the kinetics and analog specificity for binding of thyroidhormone to the plasma membrane have been repeatedly reported, a cellsurface receptor that accounts for these TR-dependent actions forthyroid hormone has not been previously identified.

Our laboratory has shown in the CV-1 monkey fibroblast cell line, whichlacks functional TR, and in other cells that T4 activates themitogen-activated protein kinase (MAPK; ERK112) signaling cascade andpromotes the phosphorylation and nuclear translocation of MAPK as earlyas 10 min following application of a physiological concentration of T4.In nuclear fractions of thyroid hormone-treated cells, we have describedcomplexes of activated MAPK and transactivator nucleoproteins that aresubstrates for the serine kinase activity of MAPK. These proteinsinclude signal transducer and activator of transcription (STAT)-1α,STAT3, p53, estrogen receptor (ER)-α and, in cells containing TR, thenuclear thyroid hormone receptor for T3 (TRβ1). Thyroid hormone-directedMAPK-dependent phosphorylation of these proteins enhances theirtranscriptional capabilities. The effects of T4-induced MAPK activationare blocked by inhibitors of the MAPK signal transduction pathway and bytetraiodothyroacetic acid (tetrac), a thyroid hormone analog whichinhibits Tq binding to the cell surface. Thyroid hormone-activated MAPKmay also act locally at the plasma membrane, e.g., on the N⁺/H⁺antiporter, rather than when translocated to the cell nucleus. A cellsurface receptor for T4, that is linked to activation of the MAPKcascade has not previously been identified.

Integrins are a family of transmembrane glycoproteins that formnoncovalent heterodimers. Extracellular domains of the integrinsinteract with a variety of ligands, including extracellular matrixglycoproteins, and the intracellular domain is linked to thecytoskeleton. Thyroid hormone was shown a decade ago to influence theinteraction of integrin with the extracellular matrix protein, laminin,but the mechanism was not known. Integrin αVβ3 has a large number ofextracellular protein ligands, including growth factors, and uponligand-binding can activate the MAPK cascade. Several of the integrinscontain an Arg-Gly-Asp (“RGD”) recognition site that is important to theliganding of matrix and other extracellular proteins that contain anArg-Gly-Asp sequence.

Thus, it would be desirable to identify and provide an initiation sitefor the induction of MAPK signaling cascades in cells treated withthyroid hormones, or analogs and polymers thereof, thereby providing formethods of modulating growth factors and other polypeptides whose cellsurface receptors clustered around this initiation site.

It is estimated that five million people are afflicted with chronicstable angina in the United States. Each year 200,000 people under theage of 65 die with what is termed “premature ischemic heart disease.”Despite medical therapy, many go on to suffer myocardial infarction anddebilitating symptoms prompting the need for revascularization witheither percutaneous transluminal coronary angioplasty or coronary arterybypass surgery. It has been postulated that one way of relievingmyocardial ischemia would be to enhance coronary collateral circulation.

Correlations have now been made between the anatomic appearance ofcoronary collateral vessels (“collaterals”) visualized at the time ofintracoronary thrombolitic therapy during the acute phase of myocardialinfarction and the creatine kinase time-activity curve, infarct size,and aneurysm formation. These studies demonstrate a protective role ofcollaterals in hearts with coronary obstructive disease, showing smallerinfarcts, less aneurysm formation, and improved ventricular functioncompared with patients in whom collaterals were not visualized. When thecardiac myocyte is rendered ischemic, collaterals develop actively bygrowth with DNA replication and mitosis of endothelial and smooth musclecells. Once ischemia develops, these factors are activated and becomeavailable for receptor occupation, which may initiate angiogenesis afterexposure to exogenous heparin. Unfortunately, the “natural” process bywhich angiogenesis occurs is inadequate to reverse the ischemia inalmost all patients with coronary artery disease.

During ischemia, adenosine is released through the breakdown of ATP.Adenosine participates in many cardio-protective biological events.Adenosine has a role in hemodynamic changes such as bradycardia andvasodilation, and adenosine has been suggested to have a role in suchunrelated phenomena as preconditioning and possibly the reduction inreperfusion injury (Ely and Beme, Circulation, 85: 893 (1992).

Angiogenesis is the development of new blood vessels from preexistingblood vessels (Mousa, S. A., In Angiogenesis Inhibitors and Stimulators:Potential Therapeutic Implications, Landes Bioscience, Georgetown, Tex.;Chapter 1, (2000)). Physiologically, angiogenesis ensures properdevelopment of mature organisms, prepares the womb for egg implantation,and plays a key role in wound healing. The development of vascularnetworks during embryogenesis or normal and pathological angiogenesisdepends on growth factors and cellular interactions with theextracellular matrix (Breier et al., Trends in Cell Biology 6:454-456(1996); Folkman, Nature Medicine 1:27-31 (1995); Risau, Nature386:671-674 (1997). Blood vessels arise during embryogenesis by twoprocesses: vasculogenesis and angiogenesis (Blood et al., Bioch.Biophys. Acta 1032:89-118 (1990). Angiogenesis is a multi-step processcontrolled by the balance of pro- and anti-angiogenic factors. Thelatter stages of this process involve proliferation and the organizationof endothelial cells (EC) into tube-like structures. Growth factors suchas FGF2 and VEGF are thought to be key players in promoting endothelialcell growth and differentiation.

Control of angiogenesis is a complex process involving local release ofvascular growth factors (P Carmeliet, Ann NY Acad Sci 902:249-260,2000), extracellular matrix, adhesion molecules and metabolic factors (RJ Tomanek, G C Schatteman, Anat Rec 261:126-135, 2000). Mechanicalforces within blood vessels may also play a role (O Hudlicka, Molec CellBiochem 147:57-68, 1995). The principal classes of endogenous growthfactors implicated in new blood vessel growth are the fibroblast growthfactor (FGF) family and vascular endothelial growth factor (VEGF) (GPages, Ann NY Acad Sci 902:187-200, 2000). The mitogen-activated proteinkinase (MAPK; ERK1/2) signal transduction cascade is involved both inVEGF gene expression and in control of proliferation of vascularendothelial cells.

Intrinsic adenosine may facilitate the coronary flow response toincreased myocardial oxygen demands and so modulate the coronary flowreserve (Ethier et al., Am. J. Physiol., H131 (1993) demonstrated thatthe addition of physiological concentrations of adenosine to humanumbilical vein endothelial cell cultures stimulates proliferation,possibly via a surface receptor. Adenosine may be a factor for humanendothelial cell growth and possibly angiogenesis. Angiogenesis appearsto be protective for patients with obstructive blood flow such ascoronary artery disease (“CAD”), but the rate at which blood vesselsgrow naturally is inadequate to reverse the disease. Thus, strategies toenhance and accelerate the body's natural angiogenesis potential shouldbe beneficial in patients with CAD.

Similarly, wound healing is a major problem in many developing countriesand diabetics have impaired wound healing and chronic inflammatorydisorders, with increased use of various cyclooxygenase-2 (CoX2)inhibitors. Angiogenesis is necessary for wound repair since the newvessels provide nutrients to support the active cells, promotegranulation tissue formation and facilitate the clearance of debris.Approximately 60% of the granulation tissue mass is composed of bloodvessels which also supply the necessary oxygen to stimulate repair andvessel growth. It is well documented that angiogenic factors are presentin wound fluid and promote repair while antiangiogenic factors inhibitrepair. Wound angiogenesis is a complex multi-step process. Despite adetailed knowledge about many angiogenic factors, little progress hasbeen made in defining the source of these factors, the regulatory eventsinvolved in wound angiogenesis and in the clinical use of angiogenicstimulants to promote repair. Further complicating the understanding ofwound angiogenesis and repair is the fact that the mechanisms andmediators involved in repair likely vary depending on the depth of thewound, type of wound (burn, trauma, etc.), and the location (muscle,skin, bone, etc.). The condition and age of the patient (diabetic,paraplegic, on steroid therapy, elderly vs infant, etc) can alsodetermine the rate of repair and response to angiogenic factors. The sexof the patient and hormonal status (premenopausal, post menopausal,etc.) may also influence the repair mechanisms and responses. Impairedwound healing particularly affects the elderly and many of the 14million diabetics in the United States. Because reduced angiogenesis isoften a causative agent for wound healing problems in these patientpopulations, it is important to define the angiogenic factors importantin wound repair and to develop clinical uses to prevent and/or correctimpaired wound healing.

Thus, there remains a need for an effective therapy in the way ofangiogenic agents as either primary or adjunctive therapy for promotionof wound healing, coronary angiogenesis, or other angiogenic-relateddisorders, with minimum side effects. Such a therapy would beparticularly useful for patients who have vascular disorders such asmyocardial infarctions, stroke or peripheral artery diseases and couldbe used prophylactically in patients who have poor coronary circulation,which places them at high risk of ischemia and myocardial infarctions.

Thyroid hormones, analogs, and polymeric conjugations play importantroles in the development of the brain. Increasing evidence suggests thatthe deprivation of polymeric thyroid hormones in the early developmentalstage causes structural and functional deficits in the CNS, but theprecise mechanism underlying this remains elusive.

The mammalian nervous system comprises a peripheral nervous system (PNS)and a central nervous system (CNS, comprising the brain and spinalcord), and is composed of two principal classes of cells: neurons andglial cells. The glial cells fill the spaces between neurons, nourishingthem and modulating their function. Certain glial cells, such as Schwanncells in the PNS and oligodendrocytes in the CNS, also provide a myelinsheath that surrounds neural processes. The myelin sheath enables rapidconduction along the neuron. In the peripheral nervous system, axons ofmultiple neurons may bundle together in order to form a nerve fiber.These, in turn, may be combined into fascicles or bundles.

During development, differentiating neurons from the central andperipheral nervous systems send out axons that grow and make contactwith specific target cells. In some cases, axons must cover enormousdistances; some grow into the periphery, whereas others are confinedwithin the central nervous system. In mammals, this stage ofneurogenesis is complete during the embryonic phase of life and neuronalcells do not multiply once they have fully differentiated.

A host of neuropathies have been identified that affect the nervoussystem. The neuropathies, which may affect neurons themselves orassociated glial cells, may result from cellular metabolic dysfunction,infection, exposure to toxic agents, autoimmunity, malnutrition, orischemia. In some cases, the cellular neuropathy is thought to inducecell death directly. In other cases, the neuropathy may inducesufficient tissue necrosis to stimulate the body's immune/inflammatorysystem and the immune response to the initial injury then destroysneural pathways.

Where the damaged neural pathway results from CNS axonal damage,autologous peripheral nerve grafts have been used to bridge lesions inthe central nervous system and to allow axons to make it back to theirnormal target area. In contrast to CNS neurons, neurons of theperipheral nervous system can extend new peripheral processes inresponse to axonal damage. This regenerative property of peripheralnervous system axons is thought to be sufficient to allow grafting ofthese segments to CNS axons. Successful grafting appears to be limited,however, by a number of factors, including the length of the CNS axonallesion to be bypassed, and the distance of the graft sites from the CNSneuronal cell bodies, with successful grafts occurring near the cellbody.

Within the peripheral nervous system, this cellular regenerativeproperty of neurons has limited ability to repair function to a damagedneural pathway. Specifically, the new axons extend randomly, and areoften misdirected, making contact with inappropriate targets that cancause abnormal function. For example, if a motor nerve is damaged,regrowing axons may contact the wrong muscles, resulting in paralysis.In addition, where severed nerve processes result in a gap of longerthan a few millimeters, e.g., greater than 10 millimeters (mm),appropriate nerve regeneration does not occur, either because theprocesses fail to grow the necessary distance, or because of misdirectedaxonal growth. Efforts to repair peripheral nerve damage by surgicalmeans has met with mixed results, particularly where damage extends overa significant distance. In some cases, the suturing steps used to obtainproper alignment of severed nerve ends stimulates the formulation ofscar tissue which is thought to inhibit axon regeneration. Even wherescar tissue formation has been reduced, as with the use of nerveguidance channels or other tubular prostheses, successful regenerationgenerally still is limited to nerve damage of less than 10 millimetersin distance. In addition, the reparative ability of peripheral neuronsis significantly inhibited where an injury or neuropathy affects thecell body itself or results in extensive degeneration of a distal axon.

Mammalian neural pathways also are at risk due to damage caused byneoplastic lesions. Neoplasias of both the neurons and glial cells havebeen identified. Transformed cells of neural origin generally lose theirability to behave as normal differentiated cells and can destroy neuralpathways by loss of function. In addition, the proliferating tumors mayinduce lesions by distorting normal nerve tissue structure, inhibitingpathways by compressing nerves, inhibiting cerbrospinal fluid or bloodsupply flow, and/or by stimulating the body's immune response.Metastatic tumors, which are a significant cause of neoplastic lesionsin the brain and spinal cord, also similarly may damage neural pathwaysand induce neuronal cell death.

One type of morphoregulatory molecule associated with neuronal cellgrowth, differentiation and development is the cell adhesion molecule(“CAM”), most notably the nerve cell adhesion molecule (N-CAM). The CAMsare members the immunoglobulin super-family. They mediate cell-cellinteractions in developing and adult tissues through homophilic binding,i.e., CAM-CAM binding on apposing cells. A number of different CAMs havebeen identified. Of these, the most thoroughly studied are N-CAM andL-CAM (liver cell adhesion molecules), both of which have beenidentified on all cells at early stages of development, as well as indifferent adult tissues. In neural tissue development, N-CAM expressionis believed to be important in tissue organization, neuronal migration,nerve-muscle tissue adhesion, retinal formation, synaptogenesis, andneural degeneration. Reduced N-CAM expression also is thought to beassociated with nerve dysfunction. For example, expression of at leastone form of N-CAM, N-CAM-180, is reduced in a mouse demyelinatingmutant. Bhat, Brain Res. 452: 373-377 (1988). Reduced levels of N-CAMalso have been associated with normal pressure hydrocephalus, Werdelin,Acta Neurol. Scand. 79: 177-181 (1989), and with type II schizophrenia.Lyons, et al., Biol. Psychiatry 23: 769-775 (1988). In addition,antibodies against N-CAM have been shown to disrupt functional recoveryin injured nerves. Remsen, Exp. Neurobiol. 110: 268-273 (1990).

Currently no satisfactory method exists to repair the damage caused bytraumatic injuries of motor neurons and diseases of motor neurons. Thereare 15,000 to 18,000 new cases of spinal cord injury each year in theUnited States. In addition, there are approximately 200,000 survivors ofspinal cord injury. The annual cost of care for these patients exceeds$7 billion. The pathophysiology following acute spinal cord trauma is acomplex and not fully understood mechanism. The primary tissue damagecaused by mechanical trauma occurs immediately and is irreversible.Allen, J. Am. Med. Assoc. 57: 878-880 (1911). Experimental evidenceindicates that much of the post-traumatic tissue damage is the result ofa reactive process that begins within minutes after the injury andcontinues for days or weeks. Janssen, et al., Spine 14: 23-32 (1989) andPanter, et al., (1992). This progressive, self-destructive processincludes pathophysiological mechanisms such as hemorrhage,post-traumatic ischemia, edema, axonal and neuronal necrosis, anddemyelinization followed by cyst formation and infarction. For review,see Tator, et al., J. Neurosurg, 75: 15-26 (1991) and Faden, Crit. Rev.Neurobiol. 7: 175-186 (1993). Proposed injurious factors includeelectrolyte changes whereby increased intracellular calcium initiates acascade of events (Young, J. Neurotrauma 9, Suppl. 1: S9-S25 (1992) andYoung, J. Emerg. Med. 11: 13-22 (1993)), biochemical changes withuncontrolled transmitter release (Liu, et al., Cell 66: 807-815 (1991)and Yanase, et al., J. Neurosurg 83: 884-888 (1995), arachidonic acidrelease, free-radical production, lipid peroxidation (Braughler, et al.,J. Neurotrauma 9, Suppl. 1: S1-S7 (1992), eicosanoid production(Demediuk, et al., J. Neurosci. Res. 20: 115-121 (1988), endogenousopioids (Faden, et al., Ann Neurol. 17: 386-390 (1985), metabolicchanges including alterations in oxygen and glucose (Faden, Crit. Rev.Neurobiol. 7: 175-186 (1993)), inflammatory changes (Blight, J.Neurotrauma 9, Suppl. 1: S83-S91 (1992), and astrocytic edema(Kimelberg, J. Neurotrauma 9, Suppl. 1: S71-S81 (1992). For the past 400years surgical approaches including laminectomy and decompression,accompanied by fusion, have been the most commonly practiced treatmentstrategies. Hansebout, “Early Management of Acute Spinal Cord Injury”,pp. 181-196 (1982) and Janssen, et al., Spine 14: 23-32 (1989). However,these procedures have not involved the application of techniques toaugment the regenerative properties of spinal cord tissue.

A host of diseases of motor neurons have been identified, includingdemyelinating diseases, myelopathies, and diseases of motor neurons suchas amyotrophic lateral sclerosis (“ALS”). INTERNAL MEDICINE, ch. 121-123(4th ed., J. H. Stein, ed., Mosby, 1994). Multiple sclerosis (“MS”) isthe most common demyelinating disorder of the central nervous system,causing patches of sclerosis (i.e., plaques) in the brain and spinalcord. MS has protean clinical manifestations, depending upon thelocation and size of the plaque. Typical symptoms include visual loss,diplopia, nystagmus, dysarthria, weakness, paresthesias, bladderabnormalities, and mood alterations. Myriad treatments have beenproposed for this long-term variable illness. The list of proposedtreatments encompasses everything from diet to electrical stimulation toacupuncture, emotional support, and various forms of immunosupressivetherapy. None have proved to be satisfactory.

Progressive loss of lower and upper motor neurons occurs in severaldiseases (e.g., primary lateral sclerosis, spinal muscular atrophy,benign focal amyotrophy). However, ALS is the most common form of motorneuron disease. Loss of both lower and upper motor neurons occur in ALS.Symptoms include progressive skeletal muscle wasting, weakness,gasciculations, and cramping. Some cases have predominant involvement ofbrainstem motoneurons (progressive bulbar palsy). Unfortunately,treatment of motor neuron and related disease is largely supportive atthis time. INTERNAL MEDICINE, ch. 123 (4th ed., J. H. Stein, ed., Mosby,1994).

Accordingly, there is a need in the art for treatments of motor neuronsdisorders and injuries, and related deficits in neural functions. It is,therefore, an object of the present invention to provide compositionsand methods for stimulating angiogenesis, for inducing neuronaldifferentiation, and for preventing the death or degeneration ofneuronal cells.

The tyrosines are iodinated at one (monoiodotyrosine) or two(diiodotyrosine) sites and then coupled to form the active hormones(diiodotyrosine+diiodotyrosine→tetraiodothyronine [thyroxine, T₄];diiodotyrosine+monoiodotyrosine→triiodothyronine [T₃]. Another source ofT₃ within the thyroid gland is the result of the outer ring deiodinationof T₄ by a selenoenzyme: type I 5′-deiodinase (5′D-I). Thyroglobulin, aglycoprotein containing T₃ and T₄ within its matrix, is taken up fromthe follicle as colloid droplets by the thyroid cells.

Lysosomes containing proteases cleave T₃ and T₄ from thyroglobulin,resulting in release of free T₃ and T₄. The iodotyrosines(monoiodotyrosine and diiodotyrosine) are also released fromthyroglobulin, but only very small amounts reach the bloodstream. Iodineis removed from them by intracellular deiodinases, and this iodine isused by the thyroid gland.

The T₄ and T₃ released from the thyroid by proteolysis reach thebloodstream, where they are bound to thyroid hormone-binding serumproteins for transport. The major thyroid hormone-binding protein isthyroxine-binding globulin (“TBG”), which has high affinity but lowcapacity for T₄ and T₃. TBG normally accounts for about 75% of the boundhormones. Other thyroid hormone-binding proteins—primarilythyroxine-binding prealbumin, also called transthyretin (“TTR”), whichhas high affinity but low capacity for T₄, and albumin, which has lowaffinity but high capacity for T₄ and T₃-account for the remainder ofthe bound serum thyroid hormones. About 0.03% of the total serum T₄ and0.3% of the total serum T₃ are free and in equilibrium with the boundhormones. Only free T₄ and T₃ are available to the peripheral tissuesfor thyroid hormone action.

Thyroid hormones have two major physiologic effects: (1) They increaseprotein synthesis in virtually every body tissue. (T₃ and T₄ entercells, where T₃, which is derived from the circulation and fromconversion of T₄ to T₃ within the cell, binds to discrete nuclearreceptors and influences the formation of mRNA.) (2) T₃ increases O₂consumption by increasing the activity of the Na⁺, K⁺-ATPase (Na pump),primarily in tissues responsible for basal O₂ consumption (ie, liver,kidney, heart, and skeletal muscle). The increased activity of Na⁺,K⁺-ATPase is secondary to increased synthesis of this enzyme; therefore,the increased O₂ consumption is also probably related to the nuclearbinding of thyroid hormones. However, a direct effect of T₃ on themitochondrion has not been ruled out. T₃ is believed to be the activethyroid hormone, although T₄ itself may be biologically active.

The pool of thyroid hormones critical for the biological actions of thehormones is the pool of free thyroid hormone. The size of this pool isdetermined for short time periods by uptake/release of thyroid hormonesinto/from cell and binding/release of thyroid hormones by thyroidhormone-binding proteins. Both proportions and absolute concentrationsof these proteins differ in blood plasma and cerebrospinal fluid(“CSF”). The most pronounced difference is found for transthyretin(“TTR”), which is the only thyroid hormone-binding plasma proteinsynthesized in the brain (Schreiber G, Southwell B R, Richardson S J.Hormone delivery systems to the brain-transthyretin. Exp Clin EndocrinolDiabetes. 1995; 103(2):75-80). TTR is also distinct from the other twothyroid hormone-binding plasma proteins in humans by the absence ofgenetic deficiencies. TTR gene expression was initiated during evolutionmuch earlier in the brain than in the liver. The structure of thedomains of TTR involved in thyroxine (TR) T4 binding has been completelyconserved for 350 million years. These observations point to a specialfunctional significance of TTR in the brain. It is proposed that this isthe determination of the level of free T4 in the extracellularcompartment of the brain. T4 can then be converted in the brain totriiodothyronine T3 by specific deiodinases. This T3 can interact withreceptors in the cell nuclei, regulating gene transcription.

Alzheimer's disease is a severe neurodegenerative disorder, andcurrently about 4 million Americans suffer from this disease. As theaging population continues to grow, this number could reach 14 millionby the middle of next century unless a cure or prevention is found. Atpresent, there is no sensitive and specific premortem test for earlydiagnosis of this disease. Alzheimer's disease is currently diagnosedbased on the clinical observation of cognitive decline, coupled with thesystematic elimination of other possible causes of those symptoms. Theconfirmation of the clinical diagnosis of “probable Alzheimer's disease”can only be made by examination of the postmortem brain. The Alzheimer'sdisease brain is characterized by the appearance of two distinctabnormal proteinaceous deposits in regions of the brain responsible forlearning and memory (e.g., cerebral cortex and hippocampus). Thesedeposits are extracellular amyloid plaques, which are characteristic ofAlzheimer's disease, and intracellular neurofibillary tangles (“NFTs”),which can be found in other neurodegenerative disorders as well. Amyloidpeptides are typically either 40 or 42 amino acids in length (“A¹⁻⁴⁰” or“A¹⁻⁴²”, respectively) and are formed from abnormal processing of alarger membrane-associated protein of unknown function, the amyloidprecurser protein (“APP”). Oligomeric aggregates of these peptides arethought to be neurotoxic, eventually resulting in synaptic degenerationand neuronal loss. The amount of amyloid deposition roughly correlateswith the severity of symptoms at the time of death.

In the past, there have been several attempts for the design ofradiopharmaceuticals that could be used as diagnostic agents for apremortem diagnosis of Alzheimer's disease. Bomebroek et al. showed thatthe amyloid-associated protein serum amyloid P component (SAP), labeledwith ¹²³I, accumulates at low levels in the cerebral cortex, possibly invessel walls, of patients with cerebral amyloidosis (Bomebroek, M., etal., Nucl. Med. Commun. (1996), Vol. 17, pp. 929-933).

Saito et al. proposed a vector-mediated delivery of ¹²³I-labeled A¹⁻⁴⁰through the blood-brain barrier. It is reported that the iodinated A¹⁻⁴⁰binds A amyloid plaque in tissue sections (Saito, Y., et al., Proc.Natl. Acad. Sci. USA 1995, Vol. 92, pp. 10227-10231). U.S. Pat. No.5,231,000 discloses antibodies with specificity to A4 amyloidpolypeptide found in the brain of Alzheimer's disease patients. However,a method to deliver these antibodies across the blood-brain barrier hasnot been described. Zhen et al. described modifications of theamyloid-binding dye known as “Congo Red™”, and complexes of thesemodified molecules with technetium and rhenium. The complexes withradioactive ions are purported to be potential imaging agents forAlzheimer's disease (Zhen et al., J. Med. Chem. (1999), Vol. 42, pp.2805-2815). However, the potential of the complexes to cross theblood-brain barrier is limited.

A group at the University of Pennsylvania in the U.S.A. (Skovronsky, M.,et al., Proc. Natl. Acad. Sci. 2000, Vol. 97, pp. 7609-7614) hasdeveloped a fluorescently labeled derivative of Congo Red that is brainpermeable and that non-specifically binds to amyloid materials (that is,peptides in-pleated sheet conformation). This compound would need to beradiolabeled and then run through pre-clinical screens forpharmacokinetics and toxicity before clinical testing. In contrast, ourinvention utilizes derivatives of naturally occurring substances aloneor in combinations for the diagnosis, prevention, and treatment ofAlzheimer's disease. Klunk et al. reported experiments with a derivativeof Congo Red™, Chrysamine G (“CG”). It is reported that CG bindssynthetic-amyloid well in vitro, and crosses the blood-brain barrier innormal mice (Klunk et al., Neurobiol. Aging (1994), Vol. 15, No. 6, pp.691-698). Bergstrom et al. presented a compound labeled with 1231 as apotential radioligand for visualization of M1 and M2 muscarinicacetylcholine receptors in Alzheimer's disease (Bergstrom et al., Eur.J. Nucl. Med. (1999), Vol. 26, pp. 1482-1485).

Recently, it has been discovered that certain specific chemokinereceptors are upregulated in the brains of patients with Alzheimer'sdisease (Horuk, R. et al., J. Immunol. (1997), Vol. 158, pp. 2882-2890);Xia et al., J. Neuro Virol (1999), Vol. 5, pp. 32-41). In addition, ithas been recently shown that the chemokine receptor CCR1 is upregulatedin the brains of patients with advanced Alzheimer's disease and absentin normal-aged brains (Halks-Miller et al, CCR1 Immunoreactivity inAlzheimer's Disease Brains, Society for Neuroscience Meeting Abstract,#787.6, Volume 24, 1998). Antagonists to the CCR1 receptor and their useas anti-inflammatory agents are described in the PCT Published PatentApplication, WO 98/56771.

None of the above described proposals have resulted in a clinicaldevelopment of an imaging agent for the early diagnosis of Alzheimer'sdisease. Accordingly, there is still a clinical need for a diagnosticagent that could be used for a reliable and early diagnosis ofAlzheimer's disease. Additionally, the proposed strategies would also beuseful for the inhibition of amyloid plaque formation or buildup inAlzheimer patients. Accordingly, it is an object of the presentinvention to provide compositions and methods for the early diagnosis,prevention, and treatment of neurodegenerative diseases, such as, forexample Alzheimer's disease.

It is interesting to note that angiogenesis also occurs in othersituations, but which are undesirable, including solid tumour growth andmetastasis; rheumatoid arthritis; psoriasis; scleroderma; and threecommon causes of blindness—diabetic retinopathy, retrolental fibroplasiaand neovascular glaucoma (in fact, diseases of the eye are almost alwaysaccompanied by vascularization. The process of wound angiogenesisactually has many features in common with tumour angiogenesis. Thus,there are some conditions, such as diabetic retinopathy or theoccurrence of primary or metastatic tumors, where angiogenesis isundesirable. Thus, there remains a need for methods by which to inhibitthe effect of angiogenic agents for the treatment of cancers.

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery that thyroid hormone,thyroid hormone analogs, and their polymeric forms, act at the cellmembrane level and have pro-angiogenic properties that are independentof the nuclear thyroid hormone effects. Accordingly, these thyroidhormone analogs and polymeric forms (i.e., angiogenic agents) can beused to treat a variety of disorders. Similarly, the invention is alsobased on the discovery that thyroid hormone analog antagonists inhibitthe pro-angiogenic effect of such analogs, and can also be used to treata variety of disorders.

Accordingly, in one aspect the invention features methods for treating acondition amenable to treatment by promoting angiogenesis byadministering to a subject in need thereof an amount of a polymeric formof thyroid hormone, or an analog thereof, effective for promotingangiogenesis. Examples of such conditions amenable to treatment bypromoting angiogenesis are provided herein and can include occlusivevascular disease, coronary disease, erectile dysfunction, myocardialinfarction, ischemia, stroke, peripheral artery vascular disorders, andwounds.

Examples of thyroid hormone analogs are also provided herein and caninclude triiodothyronine (T3), levothyroxine (T4),3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl)-phenoxy acetic acid(GC-1), or 3,5-diiodothyropropionic acid (DITPA), tetraiodothyroaceticacid (TETRAC), and triiodothyroacetic acid (TRIAC). Additional analogsare in FIG. 20 Tables A-D. These analogs can be conjugated to polyvinylalcohol, acrylic acid ethylene co-polymer, polylactic acid, or agarose.The conjugation is via covalent or non-covalent bonds depending on thepolymer used.

In one embodiment the thyroid hormone, thyroid hormone analogs, orpolymeric forms thereof are administered by parenteral, oral, rectal, ortopical means, or combinations thereof. Parenteral modes ofadministration include, for example, subcutaneous, intraperitoneal,intramuscular, or intravenous modes, such as by catheter. Topical modesof administration can include, for example, a band-aid.

In another embodiment, the thyroid hormone, thyroid hormone analogs, orpolymeric forms thereof can be encapsulated or incorporated in amicroparticle, liposome, or polymer. The polymer can include, forexample, polyglycolide, polylactide, or co-polymers thereof. Theliposome or microparticle has a size of about less than 200 nanometers,and can be administered via one or more parenteral routes, or anothermode of administration. In another embodiment the liposome ormicroparticle can be lodged in capillary beds surrounding ischemictissue, or applied to the inside of a blood vessel via a catheter.

Thyroid hormone, thyroid hormone analogs, or polymeric forms thereofaccording to the invention can also be co-administered with one or morebiologically active substances that can include, for example, growthfactors, vasodilators, anti-coagulants, anti-virals, anti-bacterials,anti-inflammatories, immuno-suppressants, analgesics, vascularizingagents, or cell adhesion molecules, or combinations thereof. In oneembodiment, the thyroid hormone analog or polymeric form is administeredas a bolus injection prior to or post-administering one or morebiologically active substance.

Growth factors can include, for example, transforming growth factoralpha (“TGFα”), transforming growth factor beta (“TGFβ”), basicfibroblast growth factor, vascular endothelial growth factor, epithelialgrowth factor, nerve growth factor, platelet-derived growth factor, andvascular permeability factor. Vasodilators can include, for example,adenosine, adenosine derivatives, or combinations thereof.Anticoagulants include, but are not limited to, heparin, heparinderivatives, anti-factor Xa, anti-thrombin, aspirin, clopidgrel, orcombinations thereof.

In another aspect of the invention, methods are provided for promotingangiogenesis along or around a medical device by coating the device witha thyroid hormone, thyroid hormone analog, or polymeric form thereofaccording to the invention prior to inserting the device into a patient.The coating step can further include coating the device with one or morebiologically active substance, such as, but not limited to, a growthfactor, a vasodilator, an anti-coagulant, or combinations thereof.Examples of medical devices that can be coated with thyroid hormoneanalogs or polymeric forms according to the invention include stents,catheters, cannulas or electrodes.

In a further aspect, the invention provides methods for treating acondition amenable to treatment by inhibiting angiogenesis byadministering to a subject in need thereof an amount of ananti-angiogenesis agent effective for inhibiting angiogenesis. Examplesof the conditions amenable to treatment by inhibiting angiogenesisinclude, but are not limited to, primary or metastatic tumors, diabeticretinopathy, and related conditions. Examples of the anti-angiogenesisagents used for inhibiting angiogenesis are also provided by theinvention and include, but are not limited to, tetraiodothyroacetic acid(TETRAC), triiodothyroacetic acid (TRIAC), monoclonal antibody LM609, XT199 or combinations thereof. Such anti-angiogenesis agents can act atthe cell surface to inhibit the pro-angiogenesis agents.

In one embodiment, the anti-angiogenesis agent is administered by aparenteral, oral, rectal, or topical mode, or combination thereof. Inanother embodiment, the anti-angiogenesis agent can be co-administeredwith one or more anti-angiogenesis therapies or chemotherapeutic agents.

In yet a further aspect, the invention provides compositions (i.e.,angiogenic agents) that include thyroid hormone, and analogs conjugatedto a polymer. The conjugation can be through a covalent or non-covalentbond, depending on the polymer. A covalent bond can occur through anester or anhydride linkage, for example. Examples of the thyroid hormoneanalogs are also provided by the instant invention and includelevothyroxine (T4), triiodothyronine (T3),3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl)-phenoxy acetic acid(GC-1), or 3,5-diiodothyropropionic acid (DITPA). In one embodiment, thepolymer can include, but is not limited to, polyvinyl alcohol, acrylicacid ethylene co-polymer, polylactic acid, or agarose.

In another aspect, the invention provides for pharmaceuticalformulations including the angiogenic agents according to the presentinvention in a pharmaceutically acceptable carrier. In one embodiment,the pharmaceutical formulations can also include one or morepharmaceutically acceptable excipients.

The pharmaceutical formulations according to the present invention canbe encapsulated or incorporated in a liposome, microparticle, orpolymer. The liposome or microparticle has a size of less than about 200nanometers. Any of the pharmaceutical formulations according to thepresent invention can be administered via parenteral, oral, rectal, ortopical means, or combinations thereof. In another embodiment, thepharmaceutical formulations can be co-administered to a subject in needthereof with one or more biologically active substances including, butnot limited to, growth factors, vasodilators, anti-coagulants, orcombinations thereof.

In other aspects, the present invention concerns the use of thepolymeric thyroid hormone analogs and pharmaceutical formulationscontaining said hormone, for the restoration of neuronal functions andenhancing survival of neural cells. For the purpose of the presentinvention, neuronal function is taken to mean the collectivephysiological, biochemical and anatomic mechanisms that allowdevelopment of the nervous system during the embryonic and postnatalperiods and that, in the adult animal, is the basis of regenerativemechanisms for damaged neurons and of the adaptive capability of thecentral nervous system when some parts of it degenerate and can notregenerate.

Therefore, the following processes occur in order to achieve neuronalfunction: denervation, reinnervation, synaptogenesis, synapticrepression, synaptic expansion, the sprouting of axons, neuralregeneration, development and organisation of neural paths and circuitsto replace the damaged ones. Therefore, the suitable patients to betreated with the polymeric thyroid hormone analogs or combinationsthereof according to the present invention are patients afflicted withdegenerative pathologies of the central nervous system (senile dementialike Alzheimer's disease, Parkinsonism, Huntington's chorea,cerebellar-spinal adrenoleucodystrophy), trauma and cerebral ischemia.

In a preferred embodiment, methods of the invention for treating motorneuron defects, including amyotrophic lateral sclerosis, multiplesclerosis, and spinal cord injury comprise administering a polymericthyroid hormone analog, or combinations thereof, and in combination withgrowth factors, nerve growth factors, or other pro-angiogenesis orneurogenesis factors. Spinal cord injuries include injuries resultingfrom a tumor, mechanical trauma, and chemical trauma. The same orsimilar methods are contemplated to restore motor function in a mammalhaving amyotrophic lateral sclerosis, multiple sclerosis, or a spinalcord injury. Administering one of the aforementioned polymeric thyroidhormone analogs alone or in combination with nerve growth factors orother neurogenesis factors also provides a prophylactic function. Suchadministration has the effect of preserving motor function in a mammalhaving, or at risk of having, amyotrophic lateral sclerosis, multiplesclerosis, or a spinal cord injury. Also according to the invention,polymeric thyroid hormone analogs alone or in combination with nervegrowth factors or other neurogenesis factors administration preservesthe integrity of the nigrostriatal pathway.

Specifically, methods of the invention for treating (pre- orpost-symptomatically) amyotrophic lateral sclerosis, multiple sclerosis,or a spinal cord injury comprise administering a polymeric thyroidhormone analog alone or in combination with nerve growth factors orother neurogenesis factors. In a particularly-preferred embodiment, thepolymeric thyroid hormone analog alone or in combination with nervegrowth factors or other neurogenesis factors is a soluble complex,comprising at least one polymeric thyroid hormone analog alone or incombination with nerve growth factors or other neurogenesis factors.

In one aspect, the invention features compositions and therapeutictreatment methods comprising administering to a mammal a therapeuticallyeffective amount of a morphogenic protein (“polymeric thyroid hormoneanalog alone or in combination with nerve growth factors or otherneurogenesis factors”), as defined herein, upon injury to a neuralpathway, or in anticipation of such injury, for a time and at aconcentration sufficient to maintain the neural pathway, includingrepairing damaged pathways, or inhibiting additional damage thereto.

In another aspect, the invention features compositions and therapeutictreatment methods for maintaining neural pathways. Such treatmentmethods include administering to the mammal, upon injury to a neuralpathway or in anticipation of such injury, a compound that stimulates atherapeutically effective concentration of an endogenous polymericthyroid hormone analog. These compounds are referred to herein aspolymeric thyroid hormone analogs alone or in combination with nervegrowth factors or other neurogenesis factors-stimulating agents, and areunderstood to include substances which, when administered to a mammal,act on tissue(s) or organ(s) that normally are responsible for, orcapable of, producing a polymeric thyroid hormone analog alone or incombination with nerve growth factors or other neurogenesis factorsand/or secreting a polymeric thyroid hormone analog alone or incombination with nerve growth factors or other neurogenesis factors, andwhich cause endogenous level of the polymeric thyroid hormone analogsalone or in combination with nerve growth factor or other neurogenesisfactors to be altered.

In particular, the invention provides methods for protecting neuronsfrom the tissue destructive effects associated with the body's immuneand inflammatory response to nerve injury. The invention also providesmethods for stimulating neurons to maintain their differentiatedphenotype, including inducing the redifferentiation of transformed cellsof neuronal origin to a morphology characteristic of untransformedneurons. In one embodiment, the invention provides means for stimulatingproduction of cell adhesion molecules, particularly nerve cell adhesionmolecules (“N-CAM”). The invention also provides methods, compositionsand devices for stimulating cellular repair of damaged neurons andneural pathways, including regenerating damaged dendrites or axons. Inaddition, the invention also provides means for evaluating the status ofnerve tissue, and for detecting and monitoring neuropathies bymonitoring fluctuations in polymeric thyroid hormone analogs alone or incombination with nerve growth factors or other neurogenesis factorslevels.

In one aspect of the invention, the polymeric thyroid hormone analogsalone or in combination with nerve growth factors or other neurogenesisfactors described herein are useful in repairing damaged neural pathwaysof the peripheral nervous system. In particular, polymeric thyroidhormone analogs alone or in combination with nerve growth factors orother neurogenesis factors are useful for repairing damaged neuralpathways, including transected or otherwise damaged nerve fibers.Specifically, the polymeric thyroid hormone analogs alone or incombination with nerve growth factor or other neurogenesis factorsdescribed herein are capable of stimulating complete axonal nerveregeneration, including vascularization and reformation of the myelinsheath. Preferably, the polymeric thyroid hormone analogs alone or incombination with nerve growth factors or other neurogenesis factors areprovided to the site of injury in a biocompatible, bioresorbable carriercapable of maintaining the polymeric thyroid hormone analogs alone or incombination with nerve growth factors or other neurogenesis factors atthe site and, where necessary, means for directing axonal growth fromthe proximal to the distal ends of a severed neuron. For example, meansfor directing axonal growth may be required where nerve regeneration isto be induced over an extended distance, such as greater than 10 mm.Many carriers capable of providing these functions are envisioned. Forexample, useful carriers include substantially insoluble materials orviscous solutions prepared as disclosed herein comprising laminin,hyaluronic acid or collagen, or other suitable synthetic, biocompatiblepolymeric materials such as polylactic, polyglycolic or polybutyricacids and/or copolymers thereof. A preferred carrier comprises anextracellular matrix composition derived, for example, from mousesarcoma cells.

In a particularly preferred embodiment, a polymeric thyroid hormoneanalog alone or in combination with nerve growth factors or otherneurogenesis factors is disposed in a nerve guidance channel which spansthe distance of the damaged pathway. The channel acts both as aprotective covering and a physical means for guiding growth of aneurite. Useful channels comprise a biocompatible membrane, which may betubular in structure, having a dimension sufficient to span the gap inthe nerve to be repaired, and having openings adapted to receive severednerve ends. The membrane may be made of any biocompatible, nonirritatingmaterial, such as silicone or a biocompatible polymer, such aspolyethylene or polyethylene vinyl acetate. The casing also may becomposed of biocompatible, bioresorbable polymers, including, forexample, collagen, hyaluronic acid, polylactic, polybutyric, andpolyglycolic acids. In a preferred embodiment, the outer surface of thechannel is substantially impermeable.

The polymeric thyroid hormone analogs alone or in combination with nervegrowth factors or other neurogenesis factors may be disposed in thechannel in association with a biocompatible carrier material, or it maybe adsorbed to or otherwise associated with the inner surface of thecasing, such as is described in U.S. Pat. No. 5,011,486, provided thatthe polymeric thyroid hormone analogs alone or in combination with nervegrowth factors or other neurogenesis factors is accessible to thesevered nerve ends.

In another aspect of the invention, polymeric thyroid hormone analogsalone or in combination with nerve growth factors or other neurogenesisfactors described herein are useful to protect against damage associatedwith the body's immune/inflammatory response to an initial injury tonerve tissue. Such a response may follow trauma to nerve tissue, caused,for example, by an autoimmune dysfunction, neoplastic lesion, infection,chemical or mechanical trauma, disease, by interruption of blood flow tothe neurons or glial cells, or by other trauma to the nerve orsurrounding material. For example, the primary damage resulting fromhypoxia or ischemia-reperfusion following occlusion of a neural bloodsupply, as in an embolic stroke, is believed to be immunologicallyassociated. In addition, at least part of the damage associated with anumber of primary brain tumors also appears to be immunologicallyrelated. Application of a polymeric thyroid hormone analog alone or incombination with nerve growth factors or other neurogenesis factors,either directly or systemically alleviates and/or inhibits theimmunologically related response to a neural injury. Alternatively,administration of an agent capable of stimulating the expression and/orsecretion in vivo of polymeric thyroid hormone analogs alone or incombination with nerve growth factors or other neurogenesis factorsexpression, preferably at the site of injury, may also be used. Wherethe injury is to be induced, as during surgery or other aggressiveclinical treatment, the polymeric thyroid hormone analogs alone or incombination with nerve growth factors or other neurogenesis factors oragent may be provided prior to induction of the injury to provide aneuroprotective effect to the nerve tissue at risk.

Generally, polymeric thyroid hormone analogs alone or in combinationwith nerve growth factors or other neurogenesis factors useful inmethods and compositions of the invention are dimeric proteins thatinduce morphogenesis of one or more eukaryotic (e.g., mammalian) cells,tissues or organs. Tissue morphogenesis includes de novo or regenerativetissue formation, such as occurs in a vertebrate embryo duringdevelopment. Of particular interest are polymeric thyroid hormoneanalogs alone or in combination with nerve growth factors or otherneurogenesis factors that induce tissue-specific morphogenesis at leastof bone or neural tissue. As defined herein, a polymeric thyroid hormoneanalog alone or in combination with nerve growth factor or otherneurogenesis factors comprises a pair of polypeptides that, when folded,form a dimeric protein that elicits morphogenetic responses in cells andtissues displaying thyroid receptors. That is, the polymeric thyroidhormone analogs alone or in combination with nerve growth factors orother neurogenesis factors generally induce a cascade of eventsincluding all of the following in a morphogenically permissiveenvironment: stimulating proliferation of progenitor cells; stimulatingthe differentiation of progenitor cells; stimulating the proliferationof differentiated cells; and, supporting the growth and maintenance ofdifferentiated cells. “Progenitor” cells are uncommitted cells that arecompetent to differentiate into one or more specific types ofdifferentiated cells, depending on their genomic repertoire and thetissue specificity of the permissive environment in which morphogenesisis induced. An exemplary progenitor cell is a hematopoeitic stem cell, amesenchymal stem cell, a basement epithelium cell, a neural crest cell,or the like. Further, polymeric thyroid hormone analogs alone or incombination with nerve growth factors or other neurogenesis factors candelay or mitigate the onset of senescence- or quiescence-associated lossof phenotype and/or tissue function. Still further, polymeric thyroidhormone analogs alone or in combination with nerve growth factors orother neurogenesis factors can stimulate phenotypic expression of adifferentiated cell type, including expression of metabolic and/orfunctional, e.g., secretory, properties thereof. In addition, polymericthyroid hormone analogs alone or in combination with nerve growth factoror other neurogenesis factors can induce redifferentiation of committedcells (e.g., osteoblasts, neuroblasts, or the like) under appropriateconditions. As noted above, polymeric thyroid hormone analogs alone orin combination with nerve growth factors or other neurogenesis factorsthat induce proliferation and/or differentiation at least of bone orneural tissue, and/or support the growth, maintenance and/or functionalproperties of neural tissue, are of particular interest herein.

Of particular interest are polymeric thyroid hormone analogs alone or incombination with nerve growth factors or other neurogenesis factorswhich, when provided to a specific tissue of a mammal, inducetissue-specific morphogenesis or maintain the normal state ofdifferentiation and growth of that tissue. In preferred embodiments, thepresent polymeric thyroid hormone analog alone or in combination withnerve growth factors or other neurogenesis factors induce the formationof vertebrate (e.g., avian or mammalian) body tissues, such as but notlimited to nerve, eye, bone, cartilage, bone marrow, ligament, toothdentin, periodontium, liver, kidney, lung, heart, or gastrointestinallining. Preferred methods may be carried out in the context ofdeveloping embryonic tissue, or at an aseptic, unscarred wound site inpost-embryonic tissue.

Other aspects of the invention include compositions and methods of usingthyroid hormone analogs and polymers thereof for imaging and diagnosisof neurodegenerative disorders, such as, for example, Alzheimer'sdisease. For example, in one aspect, the invention features T4 analogsthat have a high specificity for target sites when administered to asubject in vivo. Preferred T4 analogs show a target to non-target ratioof at least 4:1, are stable in vivo and substantially localized totarget within 1 hour after administration. In another aspect, theinvention features pharmaceutical compositions comprised of a linkerattached to the T4 analogs for Technetium, indium for gamma imagingusing single photon emission (“SPECT”) and with contrast agents for MRIimaging. Additionally, halogenated analogs that bind TTR can inhibit theformation of amyloid fibrils and thus can be utilized for the preventionand treatment of Alzheimer's disease. Such compounds can also be usedwith positron emission tomography (“PET”) imaging methods.

In other aspects, the invention also includes compositions and methodsfor modulating actions of growth factors and other polypeptides whosecell surface receptors are clustered around integrin αVβ3, or other cellsurface receptors containing the amino acid sequence Arg-Gly-Asp(“RGD”). Polypeptides that can be modulated include, for example,insulin, insulin-like growth factors, epidermal growth factors, andinterferon-γ.

The details of one or more embodiments of the invention have been setforth in the accompanying description below. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. Other features, objects, and advantagesof the invention will be apparent from the description and from theclaims. In the specification and the appended claims, the singular formsinclude plural references unless the context clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All patents and publicationscited in this specification are incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Effects of L-T4 and L-T3 on angiogenesis quantitated in thechick CAM assay. A, Control samples were exposed to PBS and additionalsamples to 1 nM T3 or 0.1 μmol/L T4 for 3 days. Both hormones causedincreased blood vessel branching in these representative images from 3experiments. B, Tabulation of mean ±SEM of new branches formed fromexisting blood vessels during the experimental period drawn from 3experiments, each of which included 9 CAM assays. At the concentrationsshown, T3 and T4 caused similar effects (1.9-fold and 2.5-foldincreases, respectively, in branch formation). **P<0.001 by 1-way ANOVA,comparing hormone-treated with PBS-treated CAM samples.

FIG. 2. Tetrac inhibits stimulation of angiogenesis by T4 andagarose-linked T4 (T4-ag). A, A 2.5-fold increase in blood vessel branchformation is seen in a representative CAM preparation exposed to 0.1μmol/L T4 for 3 days. In 3 similar experiments, there was a 2.3-foldincrease. This effect of the hormone is inhibited by tetrac (0.1μmol/L), a T4 analogue shown previously to inhibit plasma membraneactions of T4.13 Tetrac alone does not stimulate angiogenesis (C). B,T4-ag (0.1 μmol/L) stimulates angiogenesis 2.3-fold (2.9-fold in 3experiments), an effect also blocked by tetrac. C, Summary of theresults of 3 experiments that examine the actions of tetrac, T4-ag, andT4 in the CAM assay. Data (means ±SEM) were obtained from 10 images foreach experimental condition in each of 3 experiments. **P<0.001 byANOVA, comparing T4-treated and T4-agarose-treated samples withPBS-treated control samples.

FIG. 3. Comparison of the proangiogenic effects of FGF2 and T4. A,Tandem effects of T4 (0.05 μmol/L) and FGF2 (0.5 μg/mL) in submaximalconcentrations are additive in the CAM assay and equal the level ofangiogenesis seen with FGF2 (1 μg/mL in the absence of T4). B, Summaryof results from 3 experiments that examined actions of FGF2 and T4 inthe CAM assay (means ±SEM) as in A. *P<0.05; **P<0.001, comparingresults of treated samples with those of PBS-treated control samples in3 experiments.

FIG. 4. Effect of anti-FGF2 on angiogenesis caused by T4 or exogenousFGF2. A, FGF2 caused a 2-fold increase in angiogenesis in the CAM modelin 3 experiments, an effect inhibited by antibody (ab) to FGF2 (8 μg).T4 also stimulated angiogenesis 1.5-fold, and this effect was alsoblocked by FGF2 antibody, indicating that the action of thyroid hormonein the CAM model is mediated by an autocrine/paracrine effect of FGF2because T4 and T3 cause FGF2 release from cells in the CAM model (Table1). We have shown previously that a nonspecific IgG antibody has noeffect on angiogenesis in the CAM assay. B, Summary of results from 3CAM experiments that studied the action of FGF2-ab in the presence ofFGF2 or T4. *P<0.01; **P<0.001, indicating significant effects in 3experiments studying the effects of thyroid hormone and FGF2 onangiogenesis and loss of these effects in the presence of antibody toFGF2.

FIG. 5. Effect of PD 98059, a MAPK (ERK1/2) signal transduction cascadeinhibitor, on angiogenesis induced by T4, T3, and FGF2. A, Angiogenesisstimulated by T4 (0.1 μmol/L) and T3 (1 nmol/L) together is fullyinhibited by PD 98059 (3 μmol/L). B, Angiogenesis induced by FGF2 (1μg/mL) is also inhibited by PD 98059, indicating that the action of thegrowth factor is also dependent on activation of the ERK1/2 pathway. Inthe context of the experiments involving T4-agarose (T4-ag) and tetrac(FIG. 2) indicating that T4 initiates its proangiogenic effect at thecell membrane, results shown in A and B are consistent with 2 rolesplayed by MAPK in the proangiogenic action of thyroid hormone: ERK1/2transduces the early signal of the hormone that leads to FGF2elaboration and transduces the subsequent action of FGF2 onangiogenesis. C, Summary of results of 3 experiments, represented by Aand B, showing the effect of PD98059 on the actions of T4 and FGF2 inthe CAM model. *P<0.01; **P<0.001, indicating results of ANOVA on datafrom 3 experiments.

FIG. 6. T4 and FGF2 activate MAPK in ECV304 endothelial cells. Cellswere prepared in M199 medium with 0.25% hormone-depleted serum andtreated with T4 (0.1 μmol/L) for 15 minutes to 6 hours. Cells wereharvested and nuclear fractions prepared as described previously.Nucleoproteins, separated by gel electrophoresis, were immunoblottedwith antibody to phosphorylated MAPK (pERK1 and pERK2, 44 and 42 kDa,respectively), followed by a second antibody linked to aluminescence-detection system. A β-actin immunoblot of nuclear fractionsserves as a control for gel loading in each part of this figure. Eachimmunoblot is representative of 3 experiments. A, T4 causes increasedphosphorylation and nuclear translocation of ERK1/2 in ECV304 cells. Theeffect is maximal in 30 minutes, although the effect remains for ≧6hours. B, ECV304 cells were treated with the ERK1/2 activation inhibitorPD 98059 (PD; 30 μmol/L) or the PKC inhibitor CGP41251 (CGP; 100 nmol/L)for 30 minutes, after which 10⁻⁷ M T4 was added for 15 minutes to cellsamples as shown. Nuclei were harvested, and this representativeexperiment shows increased phosphorylation (activation) of ERK1/2 by T4(lane 4), which is blocked by both inhibitors (lanes 5 and 6),suggesting that PKC activity is a requisite for MAPK activation by T4 inendothelial cells. C, ECV304 cells were treated with either T4 (10⁻⁷mol/L), FGF2 (10 ng/mL), or both agents for 15 minutes. The figure showspERK1/2 accumulation in nuclei with either hormone or growth factortreatment and enhanced nuclear pERK1/2 accumulation with both agentstogether.

FIG. 7. T4 increases accumulation of FGF2 cDNA in ECV304 endothelialcells. Cells were treated for 6 to 48 hours with T4 (10⁻⁷ mol/L) andFGF2 and GAPDH cDNAs isolated from each cell aliquot. The levels of FGF2cDNA, shown in the top blot, were corrected for variations in GAPDH cDNAcontent, shown in the bottom blot, and the corrected levels of FGF2 areillustrated below in the graph (mean ±SE of mean; n=2 experiments).There was increased abundance of FGF2 transcript in RNA extracted fromcells treated with T4 at all time points. *P<0.05; **P<0.01, indicatingcomparison by ANOVA of values at each time point to control value.

FIG. 8. 7 Day Chick Embryo Tumor Growth Model. Illustration of the ChickChorioallantoic Membrane (CAM) model of tumor implant.

FIG. 9. T4 Stimulates 3D Wound Healing. Photographs of human dermalfibroblast cells exposed to T4 and control, according to the 3D WoundHealing Assay described herein.

FIG. 10. T4 Dose-Dependently Increases Wound Healing, Day 3. Asindicated by the graph, T4 increases wound healing (measured byoutmigrating cells) in a dose-dependent manner between concentrations of0.1 μM and 1.0 μM. This same increase is not seen in concentrations ofT4 between 1.0 μM and 3.0 μM.

FIG. 11. Effect of unlabeled T₄ and T₃ on ^(I-125)-T₄ binding topurified integrin. Unlabeled T₄ (10⁻⁴M to 10⁻¹¹M) or T₃ (10⁻⁴M to 10⁻⁸M)were added to purified αVβ3 integrin (2 μg/sample) and allowed toincubate for 30 min. at room temperature. Two microcuries of 1-125labeled T₄ was added to each sample. The samples were incubated for 20min. at room temperature, mixed with loading dye, and run on a 5% Nativegel for 24 hrs. at 4° C. at 45 mÅ. Following electrophoresis, the gelswere wrapped in plastic wrap and exposed to film. ^(I-125)-T₄ binding topurified αVβ3 is unaffected by unlabeled T₄ in the range of 10⁻¹¹M to10⁻⁷M, but is competed out in a dose-dependent manner by unlabeled T₄ ata concentration of 10⁻⁶M. Hot T₄ binding to the integrin is almostcompletely displaced by 10⁴M unlabeled T₄. T₃ is less effective atcompeting out T₄ binding to αVβ3, reducing the signal by 11%, 16%, and28% at 10⁻⁶M, 10⁻⁵M, and 10⁻⁴M T₃, respectively.

FIG. 12. Tetrac and an RGD containing peptide, but not an RGE containingpeptide compete out T₄ binding to purified αVβ3. A) Tetrac addition topurified αVβ3 reduces ^(I-125)-labeled T₄ binding to the integrin in adose dependent manner. 10⁻⁸M tetrac is ineffective at competing out hotT₄ binding to the integrin. The association of T₄ and αVβ3 was reducedby 38% in the presence of 10⁻⁷M tetrac and by 90% with 10⁻⁵M tetrac.Addition of an RGD peptide at 10⁻⁵M competes out T₄ binding to αVβ3.Application of 10⁻⁵M and 10⁻⁴M RGE peptide, as a control for the RGDpeptide, was unable to diminish hot T₄ binding to purified αVβ3. B)Graphical representation of the tetrac and RGD data from panel A. Datapoints are shown as the mean ±S.D. for 3 independent experiments.

FIG. 13. Effects of the monoclonal antibody LM609 on T₄ binding to αVβ3.A) LM609 was added to αVβ3 at the indicated concentrations. One μg ofLM609 per sample reduces labeled T₄ binding to the integrin by 52%.Maximal inhibition of T₄ binding to the integrin is reached whenconcentrations of LM609 are 2 μg per sample and is maintained withantibody concentrations as high as 8 μg. As a control for antibodyspecificity, 10 μg/sample Cox-2 mAB and 10 μg/sample mouse IgG wereadded to αVβ3 prior to incubation with T₄ B) Graphical representation ofdata from panel A. Data points are shown as the mean ±S.D. for 3independent experiments.

FIG. 14. Effect of RGD, RGE, tetrac, and the mAB LM609 on T₄-inducedMAPK activation. A) CV-1 cells (50-70% confluency) were treated for 30min. with 10⁻⁷ M T₄ (10⁻⁷ M total concentration, 10⁻¹⁰M freeconcentration. Selected samples were treated for 16 hrs with theindicated concentrations of either an RGD containing peptide, an RGEcontaining peptide, tetrac, or LM609 prior to the addition of T₄.Nuclear proteins ere separated by SDS-PAGE and immunoblotted withanti-phospho-MAPK (pERK1/2) antibody. Nuclear accumulation of pERK1/2 isdiminished in samples treated with 10⁻⁶ M RGD peptide or higher, but notsignificantly altered in samples treated with 10⁻⁴ M RGE. pERK1/2accumulation is decreased 76% in CV1 cells treated with 10⁻⁶M tetrac,while 10⁻⁵M and higher concentrations of tetrac reduce nuclearaccumulation of pERK1/2 to levels similar to the untreated controlsamples. The monoclonal antibody to αVβ3 LM609 decrease accumulation ofactivated MAPK in the nucleus when it is applied to CV1 cultures aconcentration of 1 μg/ml. B) Graphical representation of the data forRGD, RGE, and tetrac shown in panel A. Data points represent the mean±S.D. for 3 separate experiments.

FIG. 15. Effects of siRNA to αV and p3 on T₄ induced MAPK activation.CV1 cells were transfected with siRNA (100 nM final concentration) toαV, β3, or αV and β3 together. Two days after transfection, the cellswere treated with 10⁷M T₄. A) RT-PCR was performed from RNA isolatedfrom each transfection group to verify the specificity and functionalityof each siRNA. B) Nuclear proteins from each transfection were isolatedand subjected to SDS-PAGE.

FIG. 16. Inhibitory Effect of αVβ3 mAB (LM609) on T₄-stimulatedAngiogenesis in the CAM Model. A) Samples were exposed to PBS, T₄ (0.1μM), or T₄ plus 10 mg/ml LM609 for 3 days. Angiogenesis stimulated by T₄is substantially inhibited by the addition of the αVβ3 monoclonalantibody LM609. B) Tabulation of the mean ±SEM of new branches formedfrom existing blood vessels during the experimental period. Data wasdrawn from 3 separate experiments, each containing 9 samples in eachtreatment group. C, D) Angiogenesis stimulated by T4 or FGF2 is alsoinhibited by the addition of the αVβ3 monoclonal antibody LM609 or XT199.

FIG. 17. Polymer Compositions of Thyroid Hormone Analogs—PolymerConjugation Through an Ester Linkage Using Polyvinyl Alcohol. In thispreparation commercially available polyvinyl alcohol (or relatedco-polymers) can be esterified by treatment with the acid chloride ofthyroid hormone analogs, namely the acid chloride form. Thehydrochloride salt is neutralized by the addition of triethylamine toafford triethylamine hydrochloride which can be washed away with waterupon precipitation of the thyroid hormone ester polymer form fordifferent analogs. The ester linkage to the polymer may undergohydrolysis in vivo to release the active pro-angiogenesis thyroidhormone analog.

FIG. 18. Polymer Compositions of Thyroid Hormone Analogs—PolymerConjugation Through an Anhydride Linkage Using Acrylic Acid EthyleneCo-polymer. This is similar to the previous polymer covalent conjugationhowever this time it is through an anhydride linkage that is derivedfrom reaction of an acrylic acid co-polymer. This anhydride linkage isalso susceptible to hydrolysis in vivo to release thyroid hormoneanalog. Neutralization of the hydrochloric acid is accomplished bytreatment with triethylamine and subsequent washing of the precipitatedpolyanhydride polymer with water removes the triethylamine hydrochloridebyproduct. This reaction will lead to the formation of Thyroid hormoneanalog acrylic acid co-polymer+triethylamine. Upon in vivo hydrolysis,the thyroid hormone analog will be released over time that can becontrolled plus acrylic acid ethylene Co-polymer.

FIG. 19. Polymer Compositions of Thyroid Hormone Analogs—Entrapment in aPolylactic Acid Polymer. Polylactic acid polyester polymers (PLA)undergo hydrolysis in vivo to the lactic acid monomer and this has beenexploited as a vehicle for drug delivery systems in humans. Unlike theprior two covalent methods where the thyroid hormone analog is linked bya chemical bond to the polymer, this would be a non-covalent method thatwould encapsulate the thyroid hormone analog into PLA polymer beads.This reaction will lead to the formation of Thyroid hormone analogcontaining PLA beads in water. Filter and washing will result in theformation of thyroid hormone analog containing PLA beads, which upon invivo hydrolysis will lead to the generation of controlled levels ofthyroid hormone plus lactic acid.

FIG. 20. Thyroid Hormone Analogs Capable of Conjugation with VariousPolymers. A-D show substitutions required to achieve various thyroidhormone analogs which can be conjugated to create polymeric forms ofthyroid hormone analogs of the invention.

FIG. 21. In vitro 3-D Angiogenesis Assay FIG. 21 is a protocol andillustration of the three-dimensional in vitro sprouting assay for humanmicro-vascular endothelial on fibrin-coated beads.

FIG. 22. In Vitro Sprout Angiogenesis of HOMEC in 3-D Fibrin FIG. 22 isan illustration of human micro-vascular endothelial cell sprouting inthree dimensions under different magnifications

FIGS. 23A-E. Release of platelet-derived wound healing factors in thepresence of low level collagen

FIGS. 24A-B. Unlabeled T4 and T3 displace [¹²⁵I]-T4 from purifiedintegrin. Unlabeled T4 (10⁻¹¹ M to 10⁻⁴ M) or T3 (10⁻⁸ to 10⁻⁴ M) wereadded to purified αVβ3 integrin (2 μg/sample) prior to the addition of[¹²⁵I]-T4. (a) [¹²⁵I]-T4 binding to purified αVβ3 was unaffected byunlabeled T4 in the range of 10⁻¹¹ M to 10⁻⁷ M, but was displaced in aconcentration-dependent manner by unlabeled T4 at concentrations≧10⁻⁶ M.T3 was less effective at displacing T4 binding to αVβ3. (b) Graphicpresentation of the T4 and T3 data shows the mean ±S.D. of 3 independentexperiments.

FIGS. 25A-B. Tetrac and an RGD-containing peptide, but not anRGE-containing peptide, displace T4 binding to purified αVβ3. (a)Pre-incubation of purified αVβ3 with tetrac or an RGD-containing peptidereduced the interaction between the integrin and [¹²⁵I]-T4 in adose-dependent manner. Application of 10⁻⁵ M and 10⁻⁴ M RGE peptide, ascontrols for the RGD peptide, did not diminish labeled T4 binding topurified αVβ3. (b) Graphic presentation of the tetrac and RGD dataindicates the mean ±S.D. of results from 3 independent experiments.

FIGS. 26A-B. Integrin antibodies inhibit T4 binding to αVβ3. Theantibodies LM609 and SC7312 were added to αVβ3 at the indicatedconcentrations (μg/ml) 30 min prior to the addition of [¹²⁵I]-T4.Maximal inhibition of T4 binding to the integrin was reached when theconcentration of LM609 was 2 μg/ml and was maintained with antibodyconcentrations as high as 8 μg/ml. SC7312 reduced T4 binding to αVβ3 by46% at 2 μg/ml antibody/sample and by 58% when 8 μg/ml of antibody werepresent. As a control for antibody specificity, 10 μg/ml of anti-αVβ3mAb (P1F6) and 10 μg/ml mouse IgG were added to αVβ3 prior to incubationwith T4. The graph shows the mean ±S.D. of data from 3 independentexperiments.

FIGS. 27A-B. Effect of RGD and RGE peptides, tetrac, and the mAb LM609on T4-induced MAPK activation. (a) Nuclear accumulation of pERK1/2 wasdiminished in samples treated with 10⁻⁶ M RGD peptide or higher, but notsignificantly altered in samples treated with up to 10⁻⁴ M RGE. pERK1/2accumulation in CV-1 cells treated with 10⁻⁵ M tetrac and T4 weresimilar to levels observed in the untreated control samples. LM609, amonoclonal antibody to αVβ3, decreased accumulation of activated MAPK inthe nucleus when it was applied to CV-1 cultures in a concentration of 1μg/ml. (b) The graph shows the mean ±S.D. of data from 3 separateexperiments. Immunoblots with α-tubulin antibody are included asgel-loading controls.

FIGS. 28A-B. Effects of siRNA to αV and β3 on T4-induced MAPKactivation. CV-1 cells were transfected with siRNA (100 nM finalconcentration) to αV, β3, or αV and β3 together. Two days aftertransfection, the cells were treated with 10-7 M T4 or the vehiclecontrol for 30 min. (a) RT-PCR was performed with RNA isolated from eachtransfection group to verify the specificity and functionality of eachsiRNA. (b) Nuclear proteins from each set of transfected cells wereisolated, subjected to SDS-PAGE, and probed for pERK1/2 in the presenceor absence of treatment with T4. In the parental cells and in thosetreated with scrambled siRNA, nuclear accumulation of pERK1/2 with T4was evident. Cells treated with siRNA to αV or β3 showed an increase inpERK1/2 in the absence of T4, and a decrease with T4 treatment. Cellscontaining αV and β3 siRNAs did not respond to T4 treatment.

FIGS. 29A-B. Inhibitory effect of αVβ3 mAb (LM609) on T4-stimulatedangiogenesis in the CAM model. CAMS were exposed to filter disks treatedwith PBS, T4 (10-7 M), or T4 plus 10 μg/ml LM609 for 3 days. (a)Angiogenesis stimulated by T4 was substantially inhibited by theaddition of the αVβ3 monoclonal antibody LM609. (b) Tabulation of themean ±SEM of new branches formed from existing blood vessels during theexperimental period is shown. ***P<0.001, comparing results ofT4/LM609-treated samples with T4-treated samples in 3 separateexperiments, each containing 9 images per treatment group. Statisticalanalysis was performed by 1-way ANOVA.

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the invention will now be moreparticularly described with references to the accompanying drawings, andas pointed out by the claims. For convenience, certain terms used in thespecification, examples and claims are collected here. Unless otherwisedefined, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention pertains.

As used herein, the term “angiogenic agent” includes any compound orsubstance that promotes or encourages angiogenesis, whether alone or incombination with another substance. Examples include, but are notlimited to, T3, T4, T3 or T4-agarose, polymeric analogs of T3, T4,3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl)-phenoxy acetic acid(GC-1), or DITPA. In contrast, the terms “anti-angiogenesis agent” oranti-angiogenic agent” refer to any compound or substance that inhibitsor discourages angiogenesis, whether alone or in combination withanother substance. Examples include, but are not limited to, TETRAC,TRIAC, XT 199, and mAb LM609.

As used herein, the term “myocardial ischemia” is defined as aninsufficient blood supply to the heart muscle caused by a decreasedcapacity of the heart vessels. As used herein, the term “coronarydisease” is defined as diseases/disorders of cardiac function due to animbalance between myocardial function and the capacity of coronaryvessels to supply sufficient blood flow for normal function. Specificcoronary diseases/disorders associated with coronary disease which canbe treated with the compositions and methods described herein includemyocardial ischemia, angina pectoris, coronary aneurysm, coronarythrombosis, coronary vasospasm, coronary artery disease, coronary heartdisease, coronary occlusion and coronary stenosis.

As used herein the term “occlusive peripheral vascular disease” (alsoknown as peripheral arterial occlusive disorder) is a vasculardisorder-involving blockage in the carotid or femoral arteries,including the iliac artery. Blockage in the femoral arteries causes painand restricted movement. A specific disorder associated with occlusiveperipheral vascular disease is diabetic foot, which affects diabeticpatients, often resulting in amputation of the foot.

As used herein the terms “regeneration of blood vessels,”“angiogenesis,” “revascularization,” and “increased collateralcirculation” (or words to that effect) are considered as synonymous. Theterm “pharmaceutically acceptable” when referring to a natural orsynthetic substance means that the substance has an acceptable toxiceffect in view of its much greater beneficial effect, while the relatedthe term, “physiologically acceptable,” means the substance hasrelatively low toxicity. The term, “co-administered” means two or moredrugs are given to a patient at approximately the same time or in closesequence so that their effects run approximately concurrently orsubstantially overlap. This term includes sequential as well assimultaneous drug administration.

“Pharmaceutically acceptable salts” refers to pharmaceuticallyacceptable salts of thyroid hormone analogs, polymeric forms, andderivatives, which salts are derived from a variety of organic andinorganic counter ions well known in the art and include, by way ofexample only, sodium, potassium, calcium, magnesium, ammonium,tetra-alkyl ammonium, and the like; and when the molecule contains abasic functionality, salts of organic or inorganic acids, such ashydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate,oxalate and the like can be used as the pharmaceutically acceptablesalt. The term also includes both acid and base addition salts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid and the like, andorganic acids such as acetic acid, propionic acid, pyruvic acid, maleicacid, malonic acid, succinic acid, fumaric acid, tartaric acid, citricacid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonicacid, p-toluenesulfonic acid, salicylic acid, and the like. Particularlypreferred salts of compounds of the invention are the monochloride saltsand the dichloride salts.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, zinc, aluminum salts and the like. Preferred inorganic saltsare the ammonium, sodium, potassium, calcium, and magnesium salts. Saltsderived from organic bases include, but are not limited to, salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine,arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine,ethylenediamine, glucosamine, methylglucamine, theobromine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

“Ureido” refers to a radical of the formula —N(H)—C(O)—NH₂.

It is understood from the above definitions and examples that forradicals containing a substituted alkyl group any substitution thereoncan occur on any carbon of the alkyl group. The compounds of theinvention, or their pharmaceutically acceptable salts, may haveasymmetric carbon atoms in their structure. The compounds of theinvention and their pharmaceutically acceptable salts may thereforeexist as single enantiomers, diastereoisomers, racemates, and mixturesof enantiomers and diastereomers. All such single enantiomers,diastereoisomers, racemates and mixtures thereof are intended to bewithin the scope of this invention. Absolute configuration of certaincarbon atoms within the compounds, if known, are indicated by theappropriate absolute descriptor R or S.

Separate enantiomers can be prepared through the use of optically activestarting materials and/or intermediates or through the use ofconventional resolution techniques, e.g., enzymatic resolution or chiralHPLC.

As used herein, the phrase “growth factors” or “neurogenesis factors”refers to proteins, peptides or other molecules having a growth,proliferative, differentiative, or trophic effect on cells of the CNS orPNS. Such factors may be used for inducing proliferation ordifferentiation and can include, for example, any trophic factor thatallows cells of the CNS or PNS to proliferate, including any moleculewhich binds to a receptor on the surface of the cell to exert a trophic,or growth-inducing effect on the cell. Preferred factors include, butare not limited to, nerve growth factor (“NGF”), epidermal growth factor(“EGF”), platelet-derived growth factor (“PDGF”), insulin-like growthfactor (“IGF”), acidic fibroblast growth factor (“aFGF” or “FGF-1”),basic fibroblast growth factor (“bFGF” or “FGF-2”), and transforminggrowth factor-alpha and -beta (“TGF-α” and “TGF-β”).

“Subject” includes living organisms such as humans, monkeys, cows,sheep, horses, pigs, cattle, goats, dogs, cats, mice, rats, culturedcells therefrom, and transgenic species thereof. In a preferredembodiment, the subject is a human. Administration of the compositionsof the present invention to a subject to be treated can be carried outusing known procedures, at dosages and for periods of time effective totreat the condition in the subject. An effective amount of thetherapeutic compound necessary to achieve a therapeutic effect may varyaccording to factors such as the age, sex, and weight of the subject,and the ability of the therapeutic compound to treat the foreign agentsin the subject. Dosage regimens can be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

“Administering” includes routes of administration which allow thecompositions of the invention to perform their intended function, e.g.,promoting angiogenesis. A variety of routes of administration arepossible including, but not necessarily limited to parenteral (e.g.,intravenous, intra-arterial, intramuscular, subcutaneous injection),oral (e.g., dietary), topical, nasal, rectal, or via slow releasingmicrocarriers depending on the disease or condition to be treated. Oral,parenteral and intravenous administration are preferred modes ofadministration. Formulation of the compound to be administered will varyaccording to the route of administration selected (e.g., solution,emulsion, gels, aerosols, capsule). An appropriate compositioncomprising the compound to be administered can be prepared in aphysiologically acceptable vehicle or carrier and optional adjuvants andpreservatives. For solutions or emulsions, suitable carriers include,for example, aqueous or alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media, sterile water, creams,ointments, lotions, oils, pastes and solid carriers. Parenteral vehiclescan include sodium chloride solution, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's or fixed oils. Intravenous vehiclescan include various additives, preservatives, or fluid, nutrient orelectrolyte replenishers (See generally, Remington's PharmaceuticalScience, 16th Edition, Mack, Ed. (1980)).

“Effective amount” includes those amounts of pro-angiogenic oranti-angiogenic compounds which allow it to perform its intendedfunction, e.g., promoting or inhibiting angiogenesis inangiogenesis-related disorders as described herein. The effective amountwill depend upon a number of factors, including biological activity,age, body weight, sex, general health, severity of the condition to betreated, as well as appropriate pharmacokinetic properties. For example,dosages of the active substance may be from about 0.01 mg/kg/day toabout 500 mg/kg/day, advantageously from about 0.1 mg/kg/day to about100 mg/kg/day. A therapeutically effective amount of the activesubstance can be administered by an appropriate route in a single doseor multiple doses. Further, the dosages of the active substance can beproportionally increased or decreased as indicated by the exigencies ofthe therapeutic or prophylactic situation.

“Pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like which arecompatible with the activity of the compound and are physiologicallyacceptable to the subject. An example of a pharmaceutically acceptablecarrier is buffered normal saline (0.15M NaCl). The use of such mediaand agents for pharmaceutically active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the therapeutic compound, use thereof in the compositions suitablefor pharmaceutical administration is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

“Additional ingredients” include, but are not limited to, one or more ofthe following: excipients; surface active agents; dispersing agents;inert diluents; granulating and disintegrating agents; binding agents;lubricating agents; sweetening agents; flavoring agents; coloringagents; preservatives; physiologically degradable compositions such asgelatin; aqueous vehicles and solvents; oily vehicles and solvents;suspending agents; dispersing or wetting agents; emulsifying agents,demulcents; buffers; salts; thickening agents; fillers; emulsifyingagents; antioxidants; antibiotics; antifungal agents; stabilizingagents; and pharmaceutically acceptable polymeric or hydrophobicmaterials. Other “additional ingredients” which may be included in thepharmaceutical compositions of the invention are known in the art anddescribed, e.g., in Remington's Pharmaceutical Sciences.

Compositions

Disclosed herein are angiogenic agents comprising thyroid hormones,analogs thereof, and polymer conjugations of the hormones and theiranalogs. The disclosed compositions can be used for promotingangiogenesis to treat disorders wherein angiogenesis is beneficial.Additionally, the inhibition of these thyroid hormones, analogs andpolymer conjugations can be used to inhibit angiogenesis to treatdisorders associated with such undesired angiogenesis. As used herein,the term “angiogenic agent” includes any compound or substance thatpromotes or encourages angiogenesis, whether alone or in combinationwith another substance. Examples include, but are not limited to, T3,T4, T3 or T4-agarose, polymeric analogs of T3, T4,3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl)-phenoxy acetic acid(GC-1), or DITPA.

Polymer conjugations are used to improve drug viability. While many oldand new therapeutics are well-tolerated, many compounds need advanceddrug discovery technologies to decrease toxicity, increase circulatorytime, or modify biodistribution. One strategy for improving drugviability is the utilization of water-soluble polymers. Variouswater-soluble polymers have been shown to modify biodistribution,improve the mode of cellular uptake, change the permeability throughphysiological barriers, and modify the rate of clearance through thebody. To achieve either a targeting or sustained-release effect,water-soluble polymers have been synthesized that contain drug moietiesas terminal groups, as part of the backbone, or as pendent groups on thepolymer chain.

Representative compositions of the present invention include thyroidhormone or analogs thereof conjugated to polymers. Conjugation withpolymers can be either through covalent or non-covalent linkages. Inpreferred embodiments, the polymer conjugation can occur through anester linkage or an anhydride linkage. An example of a polymerconjugation through an ester linkage using polyvinyl alcohol is shown inFIG. 17. In this preparation commercially available polyvinyl alcohol(or related co-polymers) can be esterified by treatment with the acidchloride of thyroid hormone analogs, including the acid chloride form.The hydrochloride salt is neutralized by the addition of triethylamineto afford triethylamine hydrochloride which can be washed away withwater upon precipitation of the thyroid hormone ester polymer form fordifferent analogs. The ester linkage to the polymer may undergohydrolysis in vivo to release the active pro-angiogenesis thyroidhormone analog.

An example of a polymer conjugation through an anhydride linkage usingacrylic acid ethylene co-polymer is shown in FIG. 18. This is similar tothe previous polymer covalent conjugation, however, this time it isthrough an anhydride linkage that is derived from reaction of an acrylicacid co-polymer. This anhydride linkage is also susceptible tohydrolysis in vivo to release thyroid hormone analog. Neutralization ofthe hydrochloric acid is accomplished by treatment with triethylamineand subsequent washing of the precipitated polyanhydride polymer withwater removes the triethylamine hydrochloride byproduct. This reactionwill lead to the formation of Thyroid hormone analog acrylic acidco-polymer+triethylamine. Upon in vivo hydrolysis, the thyroid hormoneanalog will be released over time that can be controlled plus acrylicacid ethylene Co-polymer.

Another representative polymer conjugation includes thyroid hormone orits analogs conjugated to polyethylene glycol (PEG). Attachment of PEGto various drugs, proteins and liposomes has been shown to improveresidence time and decrease toxicity. PEG can be coupled to activeagents through the hydroxyl groups at the ends of the chains and viaother chemical methods. Peg itself, however, is limited to two activeagents per molecule. In a different approach, copolymers of PEG andamino acids were explored as novel biomaterials which would retain thebiocompatibility properties of PEG, but which would have the addedadvantage of numerous attachment points per molecule and which could besynthetically designed to suit a variety of applications.

Another representative polymer conjugation includes thyroid hormone orits analogs in non-covalent conjugation with polymers. This is shown indetail in FIG. 19. A preferred non-covalent conjugation is entrapment ofthyroid hormone or analogs thereof in a polylactic acid polymer.Polylactic acid polyester polymers (PLA) undergo hydrolysis in vivo tothe lactic acid monomer and this has been exploited as a vehicle fordrug delivery systems in humans. Unlike the prior two covalent methodswhere the thyroid hormone analog is linked by a chemical bond to thepolymer, this would be a non-covalent method that would encapsulate thethyroid hormone analog into PLA polymer beads. This reaction will leadto the formation of Thyroid hormone analog containing PLA beads inwater. Filter and washing will result in the formation of thyroidhormone analog containing PLA beads, which upon in vivo hydrolysis willlead to the generation of controlled levels of thyroid hormone pluslactic acid.

Still further, compositions of the present invention include thyroidhormone analogs conjugated to retinols (e.g., retinoic acid (i.e.,Vitamin A), which bind to the thyroid hormone binding proteintransthyretin (“TTR”) and retinoic binding protein (“RBP”). Thyroidhormone analogs can also be conjugated with halogenated stilbesterols,alone or in combination with retinoic acid, for use in detecting andsuppressing amyloid plaque. These analogs combine the advantageousproperties of T4-TTR, namely, their rapid uptake and prolonged retentionin brain and amyloids, with the properties of halogen substituents,including certain useful halogen isotopes for PET imaging includingfluorine-18, iodine-123, iodine-124, iodine-131, bromine-75, bromine-76,bromine-77 and bromine-82.

Furthermore, nanotechnology can be used for the creation of usefulmaterials and structures sized at the nanometer scale. The main drawbackwith biologically active substances is fragility. Nanoscale materialscan be combined with such biologically active substances to dramaticallyimprove the durability of the substance, create localized highconcentrations of the substance and reduce costs by minimizing losses.Therefore, additional polymeric conjugations include nano-particleformulations of thyroid hormones and analogs thereof. In such anembodiment, nano-polymers and nano-particles can be used as a matrix forlocal delivery of thyroid hormone and its analogs. This will aid in timecontrolled delivery into the cellular and tissue target.

Compositions of the present invention include both thyroid hormone,analogs, and derivatives either alone or in covalent or non-covalentconjugation with polymers. Examples of representative analogs andderivatives are shown in FIG. 20, Tables A-D. Table A shows T2, T3, T4,and bromo-derivatives. Table B shows alanyl side chain modifications.Table C shows hydroxy groups, diphenyl ester linkages, andD-configurations. Table D shows tyrosine analogs.

The terms “anti-angiogenesis agent” or anti-angiogenic agent” refer toany compound or substance that inhibits or discourages angiogenesis,whether alone or in combination with another substance. Examplesinclude, but are not limited to, TETRAC, TRIAC, XT 199, and mAb LM609.

The Role of Thyroid Hormone, Analogs, and Polymeric Conjugations inPromoting Angiogenesis

The pro-angiogenic effect of thyroid hormone analogs or polymeric formsdepends upon a non-genomic initiation, as tested by the susceptibilityof the hormonal effect to reduction by pharmacological inhibitors of theMAPK signal transduction pathway. Such results indicates that anotherconsequence of activation of MAPK by thyroid hormone is new blood vesselgrowth. The latter is initiated nongenomically, but of course, requiresa consequent complex gene transcription program. The ambientconcentrations of thyroid hormone are relatively stable. The CAM model,at the time we tested it, was thyroprival and thus may be regarded as asystem, which does not reproduce the intact organism.

The availability of a chick chorioallantoic membrane (CAM) assay forangiogenesis has provided a model in which to quantitate angiogenesisand to study possible mechanisms involved in the induction by thyroidhormone of new blood vessel growth. The present application discloses apro-angiogenic effect of T₄ that approximates that in the CAM model ofFGF2 and that can enhance the action of suboptimal doses of FGF2. It isfurther disclosed that the pro-angiogenic effect of the hormone isinitiated at the plasma membrane and is dependent upon activation by T₄of the MAPK signal transduction pathway. As provided above, methods fortreatment of occlusive peripheral vascular disease and coronarydiseases, in particular, the occlusion of coronary vessels, anddisorders associated with the occlusion of the peripheral vasculatureand/or coronary blood vessels are disclosed. Also disclosed arecompositions and methods for promoting angiogenesis and/or recruitingcollateral blood vessels in a patient in need thereof. The compositionsinclude an effective amount of Thyroid hormone analogs, polymeric forms,and derivatives. The methods involve the co-administration of aneffective amount of thyroid hormone analogs, polymeric forms, andderivatives in low, daily dosages for a week or more with other standardpro-angiogenesis growth factors, vasodilators, anticoagulants,thrombolytics or other vascular-related therapies.

The CAM assay has been used to validate angiogenic activity of a varietyof growth factors and compounds believed to promote angiogenesis. Forexample, T₄ in physiological concentrations was shown to bepro-angiogenic in this in vitro model and on a molar basis to have theactivity of FGF2. The presence of PTU did not reduce the effect of T₄,indicating that de-iodination of T₄ to generate T₃ was not aprerequisite in this model. A summary of the pro-angiogenesis effects ofvarious thyroid hormone analogs is listed in Table 1.

TABLE 1 Pro-angiogenesis Effects of Various Thyroid Hormone Analogs inthe CAM Model TREATMENT ANGIOGENESIS INDEX PBS (Control)  89.4 ± 9.3DITPA (0.01 uM)  133.0 ± 11.6 DITPA (0.1 uM)  167.3 ± 12.7 DITPA (0.2mM) 117.9 ± 5.6 GC-1 (0.01 uM)  169.6 ± 11.6 GC-1 (0.1 uM) 152.7 ± 9.0T4 agarose (0.1 uM) 195.5 + 8.5 T4 (0.1 uM) 143.8 ± 7.9 FGF2 (1 ug) 155± 9 n = 8 per group

The appearance of new blood vessel growth in this model requires severaldays, indicating that the effect of thyroid hormone was wholly dependentupon the interaction of the nuclear receptor for thyroid hormone (TR)with the hormone. Actions of iodothyronines that require intranuclearcomplexing of TR with its natural ligand, T₃, are by definition,genomic, and culminate in gene expression. On the other hand, thepreferential response of this model system to T₄-rather than T₃, thenatural ligand of TR-raised the possibility that angiogenesis might beinitiated nongenomically at the plasma membrane by T₄ and culminate ineffects that require gene transcription. Non-genomic actions of T₄ havebeen widely described, are usually initiated at the plasma membrane andmay be mediated by signal transduction pathways. They do not requireintranuclear ligand of iodothyronine and TR, but may interface with ormodulate gene transcription. Non-genomic actions of steroids have alsobeen well described and are known to interface with genomic actions ofsteroids or of other compounds. Experiments carried out with T₄ andtetrac or with agarose-T₄ indicated that the pro-angiogenic effect of T₄indeed very likely was initiated at the plasma membrane. Tetrac blocksmembrane-initiated effects of T₄, but does not, itself, activate signaltransduction. Thus, it is a probe for non-genomic actions of thyroidhormone. Agarose-T₄ is thought not to gain entry to the cell interiorand has been used to examine models for possible cell surface-initiatedactions of the hormone. Investigations of the pro-angiogenic effects ofthyroid hormone in the chick chorioallantoic membrane (“CAM”) modeldemonstrate that generation of new blood vessels from existing vesselswas promoted two- to three-fold by either L-thyroxine (T₄) or3,5,3′-triiodo-L-thyronine (T₃) at 10⁻⁷-10⁻M. More interestingly,T₄-agarose, a thyroid hormone analog that does not cross the cellmembrane, produced a potent pro-angiogenesis effect comparable to thatobtained with T₃ or T₄.

In part, this invention provides compositions and methods for promotingangiogenesis in a subject in need thereof. Conditions amenable totreatment by promoting angiogenesis include, for example, occlusiveperipheral vascular disease and coronary diseases, in particular, theocclusion of coronary vessels, and disorders associated with theocclusion of the peripheral vasculature and/or coronary blood vessels,erectile dysfunction, stroke, and wounds. Also disclosed arecompositions and methods for promoting angiogenesis and/or recruitingcollateral blood vessels in a patient in need thereof. The compositionsinclude an effective amount of polymeric forms of thyroid hormoneanalogs and derivatives and an effective amount of an adenosine and/ornitric oxide donor. The compositions can be in the form of a sterile,injectable, pharmaceutical formulation that includes an angiogenicallyeffective amount of thyroid hormone-like substance and adenosinederivatives in a physiologically and pharmaceutically acceptablecarrier, optionally with one or more excipients.

Myocardial Infarction

A major reason for heart failure following acute myocardial infarctionis an inadequate response of new blood vessel formation, i.e.,angiogenesis. Thyroid hormone and its analogs are beneficial in heartfailure and stimulate coronary angiogenesis. The methods of theinvention include, in part, delivering a single treatment of a thyroidhormone analog at the time of infarction either by direct injection intothe myocardium, or by simulation of coronary injection by intermittentaortic ligation to produce transient isovolumic contractions to achieveangiogenesis and/or ventricular remodeling.

Accordingly, in one aspect the invention features methods for treatingocclusive vascular disease, coronary disease, myocardial infarction,ischemia, stroke, and/or peripheral artery vascular disorders bypromoting angiogenesis by administering to a subject in need thereof anamount of a polymeric form of thyroid hormone, or an analog thereof,effective for promoting angiogenesis.

Examples of polymeric forms of thyroid hormone analogs are also providedherein and can include triiodothyronine (T3), levothyroxine (T4),(GC-1), or 3,5-diiodothyropropionic acid (DITPA) conjugated to polyvinylalcohol, acrylic acid ethylene co-polymer, polylactic acid, or agarose.

The methods also involve the co-administration of an effective amount ofthyroid hormone-like substance and an effective amount of an adenosineand/or NO donor in low, daily dosages for a week or more. One or bothcomponents can be delivered locally via catheter. Thyroid hormoneanalogs, and derivatives in vivo can be delivered to capillary bedssurrounding ischemic tissue by incorporation of the compounds in anappropriately sized liposome or microparticle. Thyroid hormone analogs,polymeric forms and derivatives can be targeted to ischemic tissue bycovalent linkage with a suitable antibody.

The method may be used as a treatment to restore cardiac function aftera myocardial infarction. The method may also be used to improve bloodflow in patients with coronary artery disease suffering from myocardialischemia or inadequate blood flow to areas other than the heartincluding, for example, occlusive peripheral vascular disease (alsoknown as peripheral arterial occlusive disease), or erectiledysfunction.

Wound Healing

Wound angiogenesis is an important part of the proliferative phase ofhealing. Healing of any skin wound other than the most superficialcannot occur without angiogenesis. Not only does any damaged vasculatureneed to be repaired, but the increased local cell activity necessary forhealing requires an increased supply of nutrients from the bloodstream.Moreover, the endothelial cells which form the lining of the bloodvessels are important in themselves as organizers and regulators ofhealing.

Thus, angiogenesis provides a new microcirculation to support thehealing wound. The new blood vessels become clinically visible withinthe wound space by four days after injury. Vascular endothelial cells,fibroblasts, and smooth muscle cells all proliferate in coordination tosupport wound granulation. Simultaneously, re-epithelialization occursto reestablish the epithelial cover. Epithelial cells from the woundmargin or from deep hair follicles migrate across the wound andestablish themselves over the granulation tissue and provisional matrix.Growth factors such as keratinocyte growth factor (KGF) mediate thisprocess. Several models (sliding versus rolling cells) ofepithelialization exist.

As thyroid hormones regulate metabolic rate, when the metabolism slowsdown due to hypothyroidism, wound healing also slows down. The role oftopically applied thyroid hormone analogs or polymeric forms in woundhealing therefore represents a novel strategy to accelerate woundhealing in diabetics and in non-diabetics with impaired wound healingabilities. Topical administration can be in the form of attachment to aband-aid. Additionally, nano-polymers and nano-particles can be used asa matrix for local delivery of thyroid hormone and its analogs. Thiswill aid in time-controlled delivery into the cellular and tissuetarget.

Accordingly, another embodiment of the invention features methods fortreating wounds by promoting angiogenesis by administering to a subjectin need thereof an amount of a polymeric form of thyroid hormone, or ananalog thereof, effective for promoting angiogenesis. For details, seeExamples 9A and 9B.

The Role of Thyroid Hormone, Analogs, and Polymeric Conjugations inCombination with Nerve Growth Factors in Inducing and MaintainingNeuronal Cells

Contrary to traditional understanding of neural induction, the presentinvention is partly based on the unexpected finding that mechanisms thatinitiate and maintain angiogenesis are effective promoters andsustainers of neurogenesis. These methods and compositions are useful,for example, for the treatment of motor neuron injury and neuropathy intrauma, injury and neuronal disorders. This invention discloses the useof various pro-angiogenesis strategies alone or in combination withnerve growth factor or other neurogenesis factors. Pro-angiogenesisfactors include polymeric thyroid hormone analogs as illustrated herein.The polymeric thyroid hormone analogs and its polymeric conjugates aloneor in combination with other pro-angiogenesis growth factors known inthe art and with nerve growth factors or other neurogenesis factors canbe combined for optimal neurogenesis.

Disclosed are therapeutic treatment methods, compositions and devicesfor maintaining neural pathways in a mammal, including enhancingsurvival of neurons at risk of dying, inducing cellular repair ofdamaged neurons and neural pathways, and stimulating neurons to maintaintheir differentiated phenotype. Additionally, a composition containingpolymeric thyroid hormone analogs, and combinations thereof, in thepresence of anti-oxidants and/or anti-inflammatory agents demonstrateneuronal regeneration and protection.

The present invention also provides thyroid hormones, analogs, andpolymeric conjugations, alone or in combination with nerve growthfactors or other neurogenesis factors, to enhance survival of neuronsand maintain neural pathways. As described herein, polymeric thyroidhormone analogs alone or in combination with nerve growth factors orother neurogenesis factors are capable of enhancing survival of neurons,stimulating neuronal CAM expression, maintaining the phenotypicexpression of differentiated neurons, inducing the redifferentiation oftransformed cells of neural origin, and stimulating axonal growth overbreaks in neural processes, particularly large gaps in axons. Morphogensalso protect against tissue destruction associated withimmunologically-related nerve tissue damage. Finally, polymeric thyroidhormone analogs alone or in combination with nerve growth factors orother neurogenesis factors may be used as part of a method formonitoring the viability of nerve tissue in a mammal.

The present invention also provides effects of polymeric thyroidhormones on synapse formation between cultured rat cortical neurons,using a system to estimate functional synapse formation in vitro.Exposure to 10-9 M polymeric thyroid hormones, 3,5,3′-triiodothyronineor thyroxine, caused an increase in the frequency of spontaneoussynchronous oscillatory changes in intracellular calcium concentration,which correlated with the number of synapses formed. The detection ofsynaptic vesicle-associated protein synapsin I by immunocytochemical andimmunoblot analysis also confirmed that exposure to thyroxinefacilitated synapse formation. The presence of amiodarone, an inhibitorof 5′-deiodinase, or amitrole, a herbicide, inhibited the synapseformation in the presence of thyroxine. Thus, the present invention alsoprovides a useful in vitro assay system for screening of miscellaneouschemicals that might interfere with synapse formation in the developingCNS by disrupting the polymeric thyroid system.

As a general matter, methods of the present invention may be applied tothe treatment of any mammalian subject at risk of or afflicted with aneural tissue insult or neuropathy. The invention is suitable for thetreatment of any primate, preferably a higher primate such as a human.In addition, however, the invention may be employed in the treatment ofdomesticated mammals which are maintained as human companions (e.g.,dogs, cats, horses), which have significant commercial value (e.g.,goats, pigs, sheep, cattle, sporting or draft animals), which havesignificant scientific value (e.g., captive or free specimens ofendangered species, or inbred or engineered animal strains), or whichotherwise have value.

The polymeric thyroid hormone analogs alone or in combination with nervegrowth factors or other neurogenesis factors described herein enhancecell survival, particularly of neuronal cells at risk of dying. Forexample, fully differentiated neurons are non-mitotic and die in vitrowhen cultured under standard mammalian cell culture conditions, using achemically defined or low serum medium known in the art. See, forexample, Chamess, J. Biol. Chem. 26: 3164-3169 (1986) and Freese, etal., Brain Res. 521: 254-264 (1990). However, if a primary culture ofnon-mitotic neuronal cells is treated with polymeric thyroid analogalone or in combination with nerve growth factor or other neurogenesisfactors, the survival of these cells is enhanced significantly. Forexample, a primary culture of striatal basal ganglia isolated from thesubstantia nigra of adult rat brain was prepared using standardprocedures, e.g., by dissociation by trituration with pasteur pipette ofsubstantia nigra tissue, using standard tissue culturing protocols, andgrown in a low serum medium, e.g., containing 50% DMEM (Dulbecco'smodified Eagle's medium), 50% F-12 medium, heat inactivated horse serumsupplemented with penicillin/streptomycin and 4 g/l glucose. Understandard culture conditions, these cells are undergoing significant celldeath by three weeks when cultured in a serum-free medium. Cell death isevidenced morphologically by the inability of cells to remain adherentand by changes in their ultrastmctural characteristics, e.g., bychromatin clumping and organelle disintegration. Specifically, cellsremained adherent and continued to maintain the morphology of viabledifferentiated neurons. In the absence of thyroid analog alone or incombination with nerve growth factor or other neurogenesis factorstreatment, the majority of the cultured cells dissociated and underwentcell necrosis.

Dysfunctions in the basal ganglia of the substantia nigra are associatedwith Huntington's chorea and parkinsonism in vivo. The ability of thepolymeric thyroid hormone analogs alone or in combination with nervegrowth factors or other neurogenesis factors defined herein to enhanceneuron survival indicates that these polymeric thyroid hormone analogsalone or in combination with nerve growth factors or other neurogenesisfactors will be useful as part of a therapy to enhance survival ofneuronal cells at risk of dying in vivo due, for example, to aneuropathy or chemical or mechanical trauma. The present inventionfurther provides that these polymeric thyroid hormone analogs alone orin combination with nerve growth factors or other neurogenesis factorsprovide a useful therapeutic agent to treat neuropathies which affectthe striatal basal ganglia, including Huntington's chorea andParkinson's disease. For clinical applications, the polymeric thyroidhormone analogs alone or in combination with nerve growth factors orother neurogenesis factors may be administered or, alternatively, apolymeric thyroid hormone analog alone or in combination with nervegrowth factors or other neurogenesis factors-stimulating agent may beadministered.

The thyroid hormone compounds described herein can also be used fornerve tissue protection from chemical trauma. The ability of thepolymeric thyroid hormone analogs alone or in combination with nervegrowth factors or other neurogenesis factors described herein to enhancesurvival of neuronal cells and to induce cell aggregation and cell—celladhesion in redifferentiated cells, indicates that the polymeric thyroidhormone analogs alone or in combination with nerve growth factors orother neurogenesis factors will be useful as therapeutic agents tomaintain neural pathways by protecting the cells defining the pathwayfrom the damage caused by chemical trauma. In particular, the polymericthyroid hormone analogs alone or in combination with nerve growthfactors or other neurogenesis factors can protect neurons, includingdeveloping neurons, from the effects of toxins known to inhibit theproliferation and migration of neurons and to interfere with cell—celladhesion. Examples of such toxins include ethanol, one or more of thetoxins present in cigarette smoke, and a variety of opiates. The toxiceffects of ethanol on developing neurons induces the neurological damagemanifested in fetal alcohol syndrome. The polymeric thyroid hormoneanalogs alone or in combination with nerve growth factors or otherneurogenesis factors also may protect neurons from the cytotoxic effectsassociated with excitatory amino acids such as glutamate.

For example, ethanol inhibits the cell—cell adhesion effects induced inpolymeric thyroid analog alone or in combination with nerve growthfactor or other neurogenesis factors-treated NG108-15 cells whenprovided to these cells at a concentration of 25-50 mM. Half maximalinhibition can be achieved with 5-10 mM ethanol, the concentration ofblood alcohol in an adult following ingestion of a single alcoholicbeverage. Ethanol likely interferes with the homophilic binding of CAMsbetween cells, rather than their induction, as polymeric thyroid analogalone or in combination with nerve growth factor or other neurogenesisfactors-induced N-CAM levels are unaffected by ethanol. Moreover, theinhibitory effect is inversely proportional to polymeric thyroid analogalone or in combination with nerve growth factor or other neurogenesisfactors concentration. Accordingly, it is envisioned that administrationof a polymeric thyroid analog alone or in combination with nerve growthfactor or other neurogenesis factors or polymeric thyroid analog aloneor in combination with nerve growth factor or other neurogenesisfactors-stimulating agent to neurons, particularly developing neurons,at risk of damage from exposure to toxins such as ethanol, may protectthese cells from nerve tissue damage by overcoming the toxin'sinhibitory effects. The polymeric thyroid analog alone or in combinationwith nerve growth factor or other neurogenesis factors described hereinalso are useful in therapies to treat damaged neural pathways resultingfrom a neuropathy induced by exposure to these toxins.

The in vivo activities of the polymeric thyroid hormone analogs alone orin combination with nerve growth factors or other neurogenesis factorsdescribed herein also are assessed readily in an animal model asdescribed herein. A suitable animal, preferably exhibiting nerve tissuedamage, for example, genetically or environmentally induced, is injectedintracerebrally with an effective amount of a polymeric thyroid hormoneanalogs alone or in combination with nerve growth factor or otherneurogenesis factors in a suitable therapeutic formulation, such asphosphate-buffered saline, pH 7. The polymeric thyroid hormone analogsalone or in combination with nerve growth factors or other neurogenesisfactors preferably is injected within the area of the affected neurons.The affected tissue is excised at a subsequent time point and the tissueevaluated morphologically and/or by evaluation of an appropriatebiochemical marker (e.g., by polymeric thyroid hormone analogs alone orin combination with nerve growth factors or other neurogenesis factorsor N-CAM localization; or by measuring the dose-dependent effect on abiochemical marker for CNS neurotrophic activity or for CNS tissuedamage, using for example, glial fibrillary acidic protein as themarker. The dosage and incubation time will vary with the animal to betested. Suitable dosage ranges for different species may be determinedby comparison with established animal models. Presented below is anexemplary protocol for a rat brain stab model.

Briefly, male Long Evans rats, obtained from standard commercialsources, are anesthetized and the head area prepared for surgery. Thecalvariae is exposed using standard surgical procedures and a holedrilled toward the center of each lobe using a 0.035K wire, justpiercing the calvariae. 25 ml solutions containing either polymericthyroid analog alone or in combination with nerve growth factor or otherneurogenesis factors (e.g., OP-1, 25 mg) or PBS then is provided to eachof the holes by Hamilton syringe. Solutions are delivered to a depthapproximately 3 mm below the surface, into the underlying cortex, corpuscallosum and hippocampus. The skin then is sutured and the animalallowed to recover.

Three days post surgery, rats are sacrificed by decapitation and theirbrains processed for sectioning. Scar tissue formation is evaluated byimmunofluorescence staining for glial fibrillary acidic protein, amarker protein for glial scarring, to qualitatively determine the degreeof scar formation. Glial fibrillary acidic protein antibodies areavailable commercially, e.g., from Sigma Chemical Co., St. Louis, Mo.Sections also are probed with anti-OP-1 antibodies to determine thepresence of OP-1. Reduced levels of glial fibrillary acidic protein areanticipated in the tissue sections of animals treated with the polymericthyroid analog alone or in combination with nerve growth factor or otherneurogenesis factors, evidencing the ability of polymeric thyroid analogalone or in combination with nerve growth factor or other neurogenesisfactors to inhibit glial scar formation and stimulate nerveregeneration.

The Role of Thyroid Hormone, Analogs, and Polymeric Conjugations forBrain Imaging, Diagnosis, and Therapies of Neurodegenerative Diseases

The present invention relates to novel pharmaceutical andradiopharmaceuticals useful for the early diagnosis, prevention, andtreatment of neurodegenerative disease, such as, for example,Alzheimer's disease. The invention also includes novel chemicalcompounds having specific binding in a biological system and capable ofbeing used for positron emission tomography (PET), single photonemission (SPECT) imaging methods, and magnetic resonance (MRI) imagingmethods. The ability of T4 and other thyroid hormone analogs to bind tolocalized ligands within the body makes it possible to utilize suchcompounds for in situ imaging of the ligands by PET, SPECT, MRI, andsimilar imaging methods. In principle, nothing need be known about thenature of the ligand, as long as binding occurs, and such binding isspecific for a class of cells, organs, tissues or receptors of interest.

PET imaging is accomplished with the aid of tracer compounds labeledwith a positron-emitting isotope (Goodman, M. M. Clinical PositronEmission Tomography, Mosby Yearbook, 1992, K. F. Hubner et al., Chapter14). For most biological materials, suitable isotopes are few. Thecarbon isotope, ¹¹C, has been used for PET, but its short half-life of20.5 minutes limits its usefulness to compounds that can be synthesizedand purified quickly, and to facilities that are proximate to acyclotron where the precursor C¹¹ starting material is generated. Otherisotopes have even shorter half-lives. N¹³ has a half-life of 10 minutesand O¹⁵ has an even shorter half-life of 2 minutes. The emissions ofboth are more energetic than those of C¹¹. Nevertheless, PET studieshave been carried out with these isotopes (Hubner, K. F., in ClinicalPositron Emission Tomography, Mosby Year Book, 1992, K. F. Hubner, etal., Chapter 2). A more useful isotope, ¹⁸F, has a half-life of 110minutes. This allows sufficient time for incorporation into aradio-labeled tracer, for purification and for administration into ahuman or animal subject. In addition, facilities more remote from acyclotron, up to about a 200 mile radius, can make use of F¹⁸ labeledcompounds. Disadvantages of ¹⁸F are the relative scarcity of fluorinatedanalogs that have functional equivalence to naturally-occurringbiological materials, and the difficulty of designing methods ofsynthesis that efficiently utilize the starting material generated inthe cyclotron. Such starting material can be either fluoride ion orfluorine gas. In the latter case only one fluorine atom of thebimolecular gas is actually a radionuclide, so the gas is designatedF-F¹⁸. Reactions using F-F¹⁸ as starting material therefore yieldproducts having only one half the radionuclide abundance of reactionsutilizing K. F¹⁸ as starting material. On the other hand, F¹⁸ can beprepared in curie quantities as fluoride ion for incorporation into aradiopharmaceutical compound in high specific activity, theoretically1.7 Ci/nmol using carrier-free nucleophilic substitution reactions. Theenergy emission of F¹⁸ is 0.635 MeV, resulting in a relatively short,2.4 mm average positron range in tissue, permitting high resolution PETimages.

SPECT imaging employs isotope tracers that emit high energy photons(.gamma.-emitters). The range of useful isotopes is greater than forPET, but SPECT provides lower three-dimensional resolution.Nevertheless, SPECT is widely used to obtain clinically significantinformation about analog binding, localization and clearance rates. Auseful isotope for SPECT imaging is I¹²³ α-gamma.-emitter with a 13.3hour half life. Compounds labeled with I¹²³ can be shipped up to about1000 miles from the manufacturing site, or the isotope itself can betransported for on-site synthesis. Eighty-five percent of the isotope'semissions are 159 KeV photons, which is readily measured by SPECTinstrumentation currently in use. The compounds of the invention can belabeled with Technetium. Technetium-99m is known to be a usefulradionuclide for SPECT imaging. The T4 analogs of the invention arejoined to a Tc-99m metal cluster through a 4-6 carbon chain which can besaturated or possess a double or triple bond.

Use of F¹⁸ labeled compounds in PET has been limited to a few analogcompounds. Most notably, ¹⁸F-fluorodeoxyglucose has been widely used instudies of glucose metabolism and localization of glucose uptakeassociated with brain activity. ¹⁸F-L-fluorodopa and other dopaminereceptor analogs have also been used in mapping dopamine receptordistribution.

Other halogen isotopes can serve for PET or SPECT imaging, or forconventional tracer labeling. These include ⁷⁵Br, ⁷⁶Br, ⁷⁷Br and ⁸²Br ashaving usable half-lives and emission characteristics. In general, thechemical means exist to substitute any halogen moiety for the describedisotopes. Therefore, the biochemical or physiological activities of anyhalogenated homolog of the described compounds are now available for useby those skilled in the art, including stable isotope halogen homolog.Astatine can be substituted for other halogen isotypes. ²¹⁰At, forexample, emits alpha particles with a half-life of 8.3 h. Other isotopesalso emit alpha particles with reasonably useful half-lives.At-substituted compounds are therefore useful for brain therapy, wherebinding is sufficiently brain-specific.

Numerous studies have demonstrated increased incorporation ofcarbohydrates and amino acids into malignant brain cells. Thisaccumulation is associated with accelerated proliferation and proteinsynthesis of such cells. The glucose analog¹⁸F-2-fluoro-2-deoxy-D-glucose (2-FDG) has been used for distinguishinghighly malignant brain brains from normal brain tissue or benign growths(DiChiro, G. et al. (1982) Neurology (NY) 32:1323-1329. However,fluorine-18 labeled 2-FDG is not the agent of choice for detecting lowgrade brain brains because high uptake in normal tissue can mask thepresence of a brain. In addition, fluorine-18 labeled 2-FDG is not theideal radiopharmaceutical for distinguishing lung brains from infectioustissue or detecting ovarian carcinoma because of high uptake of the2-FDG radioactivity in infectious tissue and in the bladder,respectively. The naturally occurring amino acid methionine, labeledwith carbon-11, has also been used to distinguish malignant tissue fromnormal tissue. But it too has relatively high uptake in normal tissue.Moreover, the half-life of carbon-11 is only 20 minutes; therefore C11methionine can not be stored for a long period of time.

Cerebrospinal fluid (“CSF”) transthyretin (“TTR”), the main CSFthyroxine (T4) carrier protein in the rat and the human is synthesizedin the choroid plexus (“CP”). After injection of ¹²⁵I-T4 in the rat,radioactive T4 accumulates first in the CP, then in the CSF and later inthe brain (Chanoine J P, Bravennan L E. The role of transthyretin in thetransport of thyroid hormone to cerebrospinal fluid and brain. Acta Med.Austriaca. 1992; 19 Suppl 1:25-8).

Compounds of the invention provide substantially improved PET imagingfor areas of the body having amyloid protein, especially of the brain.All the available positron-emitting isotopes which could be incorporatedinto a biologically-active compound have short half-lives. The practicalutility of such labeled compounds is therefore dependent on how rapidlythe labeled compound can be synthesized, the synthetic yield and theradiochemical purity of the final product. Even the shipping time fromthe isotope source, a cyclotron facility, to the hospital or laboratorywhere PET imaging is to take place, is limited. A rough calculation ofthe useful distance is about two miles per minute of half-life. ThusC¹¹, with a half-life of 20.5 m is restricted to about a 40 mile radiusfrom a source whereas compounds labeled with F¹⁸ can be used withinabout a 200 mile radius. Further requirements of an ¹⁸F-labeled compoundare that it have the binding specificity for the receptor or targetmolecule it is intended to bind, that non-specific binding to othertargets be sufficiently low to permit distinguishing between target andnon-target binding, and that the label be stable under conditions of thetest to avoid exchange with other substances in the test environment.More particularly, compounds of the invention must display adequatebinding to the desired target while failing to bind to any comparabledegree with other tissues or cells.

A partial solution to the stringent requirements for PET imaging is toemploy .gamma-emitting isotopes in SPECT imaging. I¹²³ is a commonlyused isotopic marker for SPECT, having a half-life of 13 hours for auseful range of over 1000 miles from the site of synthesis. Compounds ofthe invention can be rapidly and efficiently labeled with I¹²³ for usein SPECT analysis as an alternative to PET imaging. Furthermore, becauseof the fact that the same compound can be labeled with either isotope,it is possible for the first time to compare the results obtained by PETand SPECT using the same tracer.

The specificity of brain binding also provides utility for I-substitutedcompounds of the invention. Such compounds can be labeled withshort-lived ¹²³I for SPECT imaging or with longer-lived ¹²⁵I forlonger-term studies such as monitoring a course of therapy. Other iodineand bromine isotopes can be substituted for those exemplified.

The compounds of the invention therefore provide improved methods forbrain imaging using PET and SPECT. The methods entail administering to asubject (which can be human or animal, for experimental and/ordiagnostic purposes) an image-generating amount of a compound of theinvention, labeled with the appropriate isotope and then measuring thedistribution of the compound by PET if F¹⁸ or other positron emitter isemployed, or SPECT if I¹²³ or other gamma emitter is employed. Animage-generating amount is that amount which is at least able to providean image in a PET or SPECT scanner, taking into account the scanner'sdetection sensitivity and noise level, the age of the isotope, the bodysize of the subject and route of administration, all such variablesbeing exemplary of those known and accounted for by calculations andmeasurements known to those skilled in the art without resort to undueexperimentation.

It will be understood that compounds of the invention can be labeledwith an isotope of any atom or combination of atoms in the structure.While F¹⁸, I¹²³, and I¹²⁵ have been emphasized herein as beingparticularly useful for PET, SPECT and tracer analysis, other uses arecontemplated including those flowing from physiological orpharmacological properties of stable isotope homolog and will beapparent to those skilled in the art.

The invention also provides for technetium (Tc) labeling via Tc adducts.Isotopes of Tc, notably Tc^(99m), have been used for brain imaging. Thepresent invention provides Tc-complexed adducts of compounds of theinvention, which are useful for brain imaging. The adducts areTc-coordination complexes joined to the cyclic amino acid by a 4-6carbon chain which can be saturated or possess a double or triple bond.Where a double bond is present, either E (trans) or Z (cis) isomers canbe synthesized, and either isomer can be employed. Synthesis isdescribed for incorporating the ^(99m)Tc isotope as a last step, tomaximize the useful life of the isotope.

The following methods were employed in procedures reported herein.¹⁸F-Fluoride was produced from a Seimens cyclotron using the ¹⁸O(p,n)¹⁸F reaction with 11 MeV protons on 95% enriched ¹⁸O water. All solventsand chemicals were analytical grade and were used without furtherpurification. Melting points of compounds were determined in capillarytubes by using a Buchi SP apparatus. Thin-layer chromatographic analysis(TLC) was performed by using 250-mm thick layers of silica gel G PF-254coated on aluminum (obtained from Analtech, Inc.). Column chromatographywas performed by using 60-200 mesh silica gel (Aldrich Co.). Infraredspectra (IR) were recorded on a Beckman 18A spectrophotometer with NaClplates. Proton nuclear magnetic resonance spectra (1H NMR) were obtainedat 300 MHz with a Nicolet high-resolution instrument.

In another aspect, the invention is directed to a method of using acompound of the invention for the manufacture of a radiopharmaceuticalfor the diagnosis of Alzheimer's disease in a human. In another aspect,the invention is directed to a method of preparing compounds of theinvention.

The compounds of the invention as described herein are the thyroidhormone analogs or other TTR binding ligands, which bind to TTR and havethe ability to pass the blood-brain barrier. The compounds are thereforesuited as in vivo diagnostic agents for imaging of Alzheimer's disease.The detection of radioactivity is performed according to well-knownprocedures in the art, either by using a gamma camera or by positronemission tomography (PET).

Preferably, the free base or a pharmaceutically acceptable salt form,e.g. a monochloride or dichloride salt, of a compound of the inventionis used in a galenical formulation as diagnostic agent. The galenicalformulation containing the compound of the invention optionally containsadjuvants known in the art, e.g. buffers, sodium chloride, lactic acid,surfactants etc. A sterilization by filtration of the galenicalformulation under sterile conditions prior to usage is possible.

The radioactive dose should be in the range of 1 to 100 mCi, preferably5 to 30 mCi, and most preferably 5 to 20 mCi per application.

Of the various aspects of the invention, certain compounds (for example,compounds disclosed in Examples 16-20) are preferred. Especiallypreferred are such compounds for use as diagnostic agents in positronemission tomography (PET).

The compounds of the present invention may be administered by anysuitable route, preferably in the form of a pharmaceutical compositionadapted to such a route, and in a dose effective to bind TTR in thebrain and thereby be detected by gamma camera or PET. Typically, theadministration is parenteral, e.g., intravenously, intraperitoneally,subcutaneously, intradermally, or intramuscularly. Intravenousadministration is preferred. Thus, for example, the invention providescompositions for parenteral administration which comprise a solution ofcontrast media dissolved or suspended in an acceptable carrier, e.g.,serum or physiological sodium chloride solution.

Aqueous carriers include water, alcoholic/aqueous solutions, salinesolutions, parenteral vehicles such as sodium chloride, Ringer'sdextrose, etc. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oil and injectable organic esters such asethyl oleate. Other pharmaceutically acceptable carriers, non-toxicexcipients, including salts, preservatives, buffers and the like, aredescribed, for instance, in REMMINGTON'S PHARMACEUTICAL SCIENCES,15.sup.th Ed. Easton: Mack Publishing Co., pp. 1405-1412 and 1461-1487(1975) and THE NATIONAL FORMULARY XIV., 14.sup.th Ed. Washington:American Pharmaceutical Association (1975). Aqueous carriers, arepreferred.

Pharmaceutical composition of this invention are produced in a mannerknown per se by suspending or dissolving the compounds of thisinvention—optionally combined with the additives customary in galenicpharmacy—in an aqueous medium and then optionally sterilizing thesuspension or solution. Suitable additives are, for example,physiologically acceptable buffers (such as, for instance,tromethamine), additions of complexing agents (e.g.,diethylenetriaminepentaacetic acid) or—if required—electrolytes, e.g.,sodium chloride or—if necessary—antioxidants, such as ascorbic acid, forexample.

If suspensions or solutions of the compounds of this invention in wateror physiological saline solution are desirable for enteraladministration or other purposes, they are mixed with one or several ofthe auxiliary agents (e.g., methylcellulose, lactose, mannitol) and/ortensides (e.g., lecithins, “Tween”, “Myrj”) and/or flavoring agents toimprove taste (e.g., ethereal oils), as customary in galenic pharmacy.

The compositions may be sterilized by conventional, well knownsterilization techniques, or may be sterile filtered. The resultingaqueous solutions may be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc.

For the compounds according to the invention having radioactivehalogens, these compounds can be shipped as “hot” compounds, i.e., withthe radioactive halogen in the compound and administered in e.g., aphysiologically acceptable saline solution. In the case of the metalcomplexes, these compounds can be shipped as “cold” compounds, i.e.,without the radioactive ion, and then mixed with Tc-generator eluate orRe-generator eluate.

The Role of Thyroid Hormone Analogs and Polymeric Conjugations inModulating the Actions of Polypeptides Whose Cell Surface Receptors areClustered Around Integrin αβ3, or Other RGD-Containing Compounds

Integrin αVβ3 is a heterodimeric plasma membrane protein with severalextracellular matrix protein ligands containing an amino acid sequenceArg-Gly-Asp (“RGD”). Using purified integrin, we discovered thatintegrin αVβ3 binds T4 and that this interaction is perturbed by αVβ3antagonists. Radioligand-binding studies revealed that purified αVβ3binds T4 with high affinity (EC50, 371 pM), and appears to bind T4preferentially over T3. This is consistent with previous reports thatshow MAPK activation and nuclear translocation, as well ashormone-induced angiogenesis, by T4, compared to T3. Integrin αVβ3antagonists inhibit binding of T4 to the integrin and, importantly,prevent activation by T4 of the MAPK signaling cascade. This functionalconsequence-MAPK activation—of hormone-binding to the integrin, togetherwith inhibition of the MAPK-dependent pro-angiogenic action of thyroidhormone by integrin αVβ3 antagonists, allow us to describe theiodothyronine-binding site on the integrin as a receptor. It should benoted that 3-iodothyronamine, a thyroid hormone derivative, has recentlybeen shown by Scanlan et al. to bind to a trace amine receptor (TAR I),but the actions of this analog interestingly are antithetic to those ofT4 and T3.

The traditional ligands of integrins are proteins. That a smallmolecule, thyroid hormone, is also a ligand of an integrin is a novelfinding. The present invention also discloses that, resveratrol, apolyphenol with some estrogenic activity, binds to integrin αVβ3 with afunctional cellular consequence, apoptosis, different from those thatresult from the binding of thyroid hormone. The site on the integrin atwhich T4 binds is at or near the RGD binding groove of the heterodimericintegrin. It is possible, however, that αVβ3 binds T4 elsewhere on theprotein and that the occupation of the RGD recognition site by tetrac orby RGD-containing peptides allosterically blocks the T4 binding site orcauses a conformational change within the integrin that renders the T4site unavailable. Accordingly, the modulation by T4 of thelaminin-integrin interaction of astrocytes may be a consequence ofbinding of the hormone to the integrin. The possibility thus exists thatat the cell exterior thyroid hormone may affect the liganding byintegrin αVβ3 of extracellular matrix proteins in addition to laminin.

Actions of T4 that are nongenomic in mechanism have been well documentedin recent years. A number of these activities are MAPK-mediated. We haveshown that initial steps in activation of the MAPK cascade by thyroidhormone, including activation of protein kinase C, are sensitive toGTPγS and pertussis toxin, indicating that the plasma membrane receptorfor thyroid hormone is G protein-sensitive. It should be noted thatcertain cellular functions mediated by integrin αVβ3 have been shown byothers to be G protein-modulated. For example, site-directed mutagenesisof the RGD binding domain abolishes the ability of the nucleotidereceptor P2Y2 to activate G₀, while the activation of G_(q), was notaffected. Wang et al. demonstrated that an integrin-associated protein,IAP/CD47, induced smooth muscle cell migration via G_(i)-mediatedinhibition of MAPK activation.

In addition to linking the binding of T4 and other analogs by integrinαVβ3 to activation of a specific intracellular signal transductionpathway, the present invention also discloses that the liganding of thehormone by the integrin is critical to induction by T4 of MAPK-dependentangiogenesis. In the CAM model, significant vessel growth occurs after48-72 h of T4 treatment, indicating that the plasma membrane effects ofT4 can result in complex transcriptional changes. Thus, what isinitiated as a nongenomic action of the hormone—transduction of the cellsurface T4 signal—interfaces with genomic effects of the hormone thatculminate in neovascularization. Interfaces of nongenomic and genomicactions of thyroid hormone have previously been described, e.g.,MAPK-dependent phosphorylation at Ser-142 of TRβ1 that is initiated atthe cell surface by T4 and that results in shedding by TR of corepressorproteins and recruitment of coactivators. The instant invention alsodiscloses that T4 stimulates growth of C-6 glial cells by aMAPK-dependent mechanism that is inhibited by RGD peptide, and thatthyroid hormone causes MAPK-mediated serine-phosphorylation of thenuclear estrogen receptor (ERα) in MCF-7 cells by a process we now knowto be inhibitable by an RGD peptide. These findings in several celllines all support the participation of the integrin in functionalresponses of cells to thyroid hormone.

Identification of αVβ3 as a membrane receptor for thyroid hormoneindicates clinical significance of the interaction of the integrin andthe hormone and the downstream consequence of angiogenesis. For example,αVβ3 is overexpressed in many tumors and this overexpression appears toplay a role in tumor invasion and growth. Relatively constantcirculating levels of thyroid hormone can facilitate tumor-associatedangiogenesis. In addition to demonstrating the pro-angiogenic action ofT4 in the CAM model here and elsewhere, the present invention alsodiscloses that human dermal microvascular endothelial cells also formnew blood vessels when exposed to thyroid hormone. Local delivery ofαVβ3 antagonists or tetrac around tumor cells might inhibit thyroidhormone-stimulated angiogenesis. Although tetrac lacks many of thebiologic activities of thyroid hormone, it does gain access to theinterior of certain cells. Anchoring of tetrac, or specific RGDantagonists, to non-immunogenic substrates (agarose or polymers) wouldexclude the possibility that the compounds could cross the plasmamembrane, yet retain as shown here the ability to prevent T4-inducedangiogenesis. The agarose-T4 used in the present studies is thus aprototype for a new family of thyroid hormone analogues that havespecific cellular effects, but do not gain access to the cell interior.

Accordingly, the Examples herein identify integrin αVβ3 as a cellsurface receptor for thyroid hormone (L-thyroxine, T4) and as theinitiation site for T4-induced activation of intracellular signalingcascades. αVβ3 dissociably binds radiolabeled T4 with high affinity;radioligand-binding is displaced by tetraiodothyroacetic acid (tetrac),αVβ3 antibodies and by an integrin RGD recognition site peptide. CV-1cells lack nuclear thyroid hormone receptor but bear plasma membraneαVβ3; treatment of these cells with physiological concentrations of T4activates the MAPK pathway, an effect inhibited by tetrac, RGD peptideand αVβ3 antibodies. Inhibitors of T4-binding to the integrin also blockthe MAPK-mediated pro-angiogenic action of T4. T4-inducedphosphorylation of MAPK is blocked by siRNA knockdown of αV and β3.These findings indicate that T4 binds to αVβ3 near the RGD recognitionsite and show that hormone-binding to αVβ3 has physiologic consequences.

The Role of Thyroid Hormone Analogs, and Polymeric Conjugations inInhibiting Angiogenesis

The invention also provides, in another part, compositions and methodsfor inhibiting angiogenesis in a subject in need thereof. Conditionsamenable to treatment by inhibiting angiogenesis include, for example,primary or metastatic tumors and diabetic retinopathy. The compositionscan include an effective amount of TETRAC, TRIAC or mAb LM609. Thecompositions can be in the form of a sterile, injectable, pharmaceuticalformulation that includes an anti-angiogenically effective amount of ananti-angiogenic substance in a physiologically and pharmaceuticallyacceptable carrier, optionally with one or more excipients.

In a further aspect, the invention provides methods for treating acondition amenable to treatment by inhibiting angiogenesis byadministering to a subject in need thereof an amount of ananti-angiogenesis agent effective for inhibiting angiogenesis.

Examples of the anti-angiogenesis agents used for inhibitingangiogenesis are also provided by the invention and include, but are notlimited to, tetraiodothyroacetic acid (TETRAC), triiodothyroacetic acid(TRIAC), monoclonal antibody LM609, or combinations thereof. Suchanti-angiogenesis agents can act at the cell surface to inhibit thepro-angiogenesis agents.

Cancer-Related New Blood Vessel Growth

Examples of the conditions amenable to treatment by inhibitingangiogenesis include, but are not limited to, primary or metastatictumors, including, but not limited to glioma and breast cancer. In sucha method, compounds which inhibit the thyroid hormone-induced angiogeniceffect are used to inhibit angiogenesis. Details of such a method isillustrated in Example 12.

Diabetic Retinopathy

Examples of the conditions amenable to treatment by inhibitingangiogenesis include, but are not limited to diabetic retinopathy, andrelated conditions. In such a method, compounds which inhibit thethyroid hormone-induced angiogenic effect are used to inhibitangiogenesis. Details of such a method is illustrated in Examples 8A andB.

It is known that proliferative retinopathy induced by hypoxia (ratherthan diabetes) depends upon alphaV (αV) integrin expression (E Chavakiset al., Diabetologia 45:262-267, 2002). It is proposed herein thatthyroid hormone action on a specific integrin alphaVbeta-3 (αVβ3) ispermissive in the development of diabetic retinopathy. Integrin αVβ3 isidentified herein as the cell surface receptor for thyroid hormone.Thyroid hormone, its analogs, and polymer conjugations, act via thisreceptor to induce angiogenesis.

Methods of Treatment and Formulations

Thyroid hormone analogs, polymeric forms, and derivatives can be used ina method for promoting angiogenesis in a patient in need thereof. Themethod involves the co-administration of an effective amount of thyroidhormone analogs, polymeric forms, and derivatives in low, daily dosagesfor a week or more. The method may be used as a treatment to restorecardiac function after a myocardial infarction. The method may also beused to improve blood flow in patients with coronary artery diseasesuffering from myocardial ischemia or inadequate blood flow to areasother than the heart, for example, peripheral vascular disease, forexample, peripheral arterial occlusive disease, where decreased bloodflow is a problem.

The compounds can be administered via any medically acceptable meanswhich is suitable for the compound to be administered, including oral,rectal, topical or parenteral (including subcutaneous, intramuscular andintravenous) administration. For example, adenosine has a very shorthalf-life. For this reason, it is preferably administered intravenously.However, adenosine A.sub.2 agonists have been developed which have muchlonger half-lives, and which can be administered through other means.Thyroid hormone analogs, polymeric forms, and derivatives can beadministered, for example, intravenously, oral, topical, intranasaladministration.

In some embodiments, the thyroid hormone analogs, polymeric forms, andderivatives are administered via different means.

The amounts of the thyroid hormone, its analogs, polymeric forms, andderivatives required to be effective in stimulating angiogenesis will,of course, vary with the individual being treated and is ultimately atthe discretion of the physician. The factors to be considered includethe condition of the patient being treated, the efficacy of theparticular adenosine A₂ receptor agonist being used, the nature of theformulation, and the patient's body weight. Occlusion-treating dosagesof thyroid hormone analogs or its polymeric forms, and derivatives areany dosages that provide the desired effect.

The compounds described above are preferably administered in aformulation including thyroid hormone analogs or its polymeric forms,and derivatives together with an acceptable carrier for the mode ofadministration. Any formulation or drug delivery system containing theactive ingredients, which is suitable for the intended use, as aregenerally known to those of skill in the art, can be used. Suitablepharmaceutically acceptable carriers for oral, rectal, topical orparenteral (including subcutaneous, intraperitoneal, intramuscular andintravenous) administration are known to those of skill in the art. Thecarrier must be pharmaceutically acceptable in the sense of beingcompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof.

Formulations suitable for parenteral administration conveniently includesterileaqueous preparation of the active compound, which is preferablyisotonic with the blood of the recipient. Thus, such formulations mayconveniently contain distilled water, 5% dextrose in distilled water orsaline. Useful formulations also include concentrated solutions orsolids containing the compound of formula (I), which upon dilution withan appropriate solvent give a solution suitable for parentaladministration above.

For enteral administration, a compound can be incorporated into an inertcarrier in discrete units such as capsules, cachets, tablets orlozenges, each containing a predetermined amount of the active compound;as a powder or granules; or a suspension or solution in an aqueousliquid or non-aqueous liquid, e.g., a syrup, an elixir, an emulsion or adraught. Suitable carriers may be starches or sugars and includelubricants, flavorings, binders, and other materials of the same nature.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active compound in a free-flowingform, e.g., a powder or granules, optionally mixed with accessoryingredients, e.g., binders, lubricants, inert diluents, surface activeor dispersing agents. Molded tablets may be made by molding in asuitable machine, a mixture of the powdered active compound with anysuitable carrier.

A syrup or suspension may be made by adding the active compound to aconcentrated, aqueous solution of a sugar, e.g., sucrose, to which mayalso be added any accessory ingredients. Such accessory ingredients mayinclude flavoring, an agent to retard crystallization of the sugar or anagent to increase the solubility of any other ingredient, e.g., as apolyhydric alcohol, for example, glycerol or sorbitol.

Formulations for rectal administration may be presented as a suppositorywith a conventional carrier, e.g., cocoa buffer or Witepsol S55(trademark of Dynamite Nobel Chemical, Germany), for a suppository base.

Alternatively, the compound may be administered in liposomes ormicrospheres (or microparticles). Methods for preparing liposomes andmicrospheres for administration to a patient are well known to those ofskill in the art. U.S. Pat. No. 4,789,734, the contents of which arehereby incorporated by reference, describes methods for encapsulatingbiological materials in liposomes. Essentially, the material isdissolved in an aqueous solution, the appropriate phospholipids andlipids added, along with surfactants if required, and the materialdialyzed or sonicated, as necessary. A review of known methods isprovided by G. Gregoriadis, Chapter 14, “Liposomes,” Drug Carriers inBiology and Medicine, pp. 2⁸7-341 (Academic Press, 1979).

Microspheres formed of polymers or proteins are well known to thoseskilled in the art, and can be tailored for passage through thegastrointestinal tract directly into the blood stream. Alternatively,the compound can be incorporated and the microspheres, or composite ofmicrospheres, implanted for slow release over a period of time rangingfrom days to months. See, for example, U.S. Pat. Nos. 4,906,474,4,925,673 and 3,625,214, and Jein, TIPS 19:155-157 (1998), the contentsof which are hereby incorporated by reference.

In one embodiment, the thyroid hormone analogs or its polymeric forms,and adenosine derivatives can be formulated into a liposome ormicroparticle, which is suitably sized to lodge in capillary bedsfollowing intravenous administration. When the liposome or microparticleis lodged in the capillary beds surrounding ischemic tissue, the agentscan be administered locally to the site at which they can be mosteffective. Suitable liposomes for targeting ischemic tissue aregenerally less than about 200 nanometers and are also typicallyunilamellar vesicles, as disclosed, for example, in U.S. Pat. No.5,593,688 to Baldeschweiler, entitled “Liposomal targeting of ischemictissue,” the contents of which are hereby incorporated by reference.

Preferred microparticles are those prepared from biodegradable polymers,such as polyglycolide, polylactide and copolymers thereof. Those ofskill in the art can readily determine an appropriate carrier systemdepending on various factors, including the desired rate of drug releaseand the desired dosage.

In one embodiment, the formulations are administered via catheterdirectly to the inside of blood vessels. The administration can occur,for example, through holes in the catheter. In those embodiments whereinthe active compounds have a relatively long half life (on the order of 1day to a week or more), the formulations can be included inbiodegradable polymeric hydrogels, such as those disclosed in U.S. Pat.No. 5,410,016 to Hubbell et al. These polymeric hydrogels can bedelivered to the inside of a tissue lumen and the active compoundsreleased over time as the polymer degrades. If desirable, the polymerichydrogels can have microparticles or liposomes which include the activecompound dispersed therein, providing another mechanism for thecontrolled release of the active compounds.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active compound intoassociation with a carrier, which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound into association with a liquidcarrier or a finely divided solid carrier and then, if necessary,shaping the product into desired unit dosage form.

The formulations can optionally include additional components, such asvarious biologically active substances such as growth factors (includingTGF-.beta., basic fibroblast growth factor (FGF2), epithelial growthfactor (EGF), transforming growth factors .alpha. and .beta. (TGF alpha.and beta.), nerve growth factor (NGF), platelet-derived growth factor(PDGF), and vascular endothelial growth factor/vascular permeabilityfactor (VEGF/VPF)), antiviral, antibacterial, anti-inflammatory,immuno-suppressant, analgesic, vascularizing agent, and cell adhesionmolecule.

In addition to the aforementioned ingredients, the formulations mayfurther include one or more optional accessory ingredient(s) utilized inthe art of pharmaceutical formulations, e.g., diluents, buffers,flavoring agents, binders, surface active agents, thickeners,lubricants, suspending agents, preservatives (including antioxidants)and the like.

Formulations and Methods of Treatment

Polymeric thyroid hormone analogs alone or in combination with nervegrowth factors or other neurogenesis factors inducers, or agonists ofpolymeric thyroid hormone analogs alone or in combination with nervegrowth factors or other neurogenesis factors receptors of the presentinvention may be administered by any route which is compatible with theparticular polymeric thyroid hormone analog alone or in combination withnerve growth factors or other neurogenesis factors, inducer, or agonistemployed. Thus, as appropriate, administration may be oral orparenteral, including intravenous and intraperitoneal routes ofadministration. In addition, administration may be by periodicinjections of a bolus of the polymeric thyroid hormone analog alone orin combination with nerve growth factors or other neurogenesis factors,inducer or agonist, or may be made more continuous by intravenous orintraperitoneal administration from a reservoir which is external (e.g.,an i.v. bag) or internal (e.g., a bioerodable implant, or a colony ofimplanted, polymeric thyroid analog alone or in combination with nervegrowth factor or other neurogenesis factors-producing cells).

Therapeutic agents of the invention (i.e., polymeric thyroid hormoneanalogs alone or in combination with nerve growth factors or otherneurogenesis factors, inducers or agonists of polymeric thyroid hormoneanalogs alone or in combination with nerve growth factors or otherneurogenesis factors receptors) may be provided to an individual by anysuitable means, directly (e.g., locally, as by injection, implantationor topical administration to a tissue locus) or systemically (e.g.,parenterally or orally). Where the agent is to be provided parenterally,such as by intravenous, subcutaneous, intramolecular, ophthalmic,intraperitoneal, intramuscular, buccal, rectal, vaginal, intraorbital,intracerebral, intracranial, intraspinal, intraventricular, intrathecal,intracisternal, intracapsular, intranasal or by aerosol administration,the agent preferably comprises part of an aqueous or physiologicallycompatible fluid suspension or solution. Thus, the polymeric thyroidhormone analogs alone or in combination with nerve growth factors orother neurogenesis factors carrier or vehicle is physiologicallyacceptable so that in addition to delivery of the desired agent to thepatient, it does not otherwise adversely affect the patient'selectrolyte and/or volume balance. The fluid medium for the agent thuscan comprise normal physiologic saline (e.g., 9.85% aqueous NaCl, 0.15M,pH 7-7.4).

Association of the dimer with a polymeric thyroid hormone analog prodomain results in the pro form of the polymeric thyroid hormone analogwhich typically is more soluble in physiological solutions than thecorresponding mature form.

Useful solutions for parenteral administration may be prepared by any ofthe methods well known in the pharmaceutical art, described, forexample, in REMINGTON'S PHARMACEUTICAL SCIENCES (Gennaro, A., ed.), MackPub., 1990. Formulations of the therapeutic agents of the invention mayinclude, for example, polyalkylene glycols such as polyethylene glycol,oils of vegetable origin, hydrogenated naphthalenes, and the like.Formulations for direct administration, in particular, may includeglycerol and other compositions of high viscosity to help maintain theagent at the desired locus. Biocompatible, preferably bioresorbable,polymers, including, for example, hyaluronic acid, collagen, tricalciumphosphate, polybutyrate, lactide, and glycolide polymers andlactide/glycolide copolymers, may be useful excipients to control therelease of the agent in vivo. Other potentially useful parenteraldelivery systems for these agents include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, andliposomes. Formulations for inhalation administration contain asexcipients, for example, lactose, or may be aqueous solutionscontaining, for example, polyoxyethylene-9-lauryl ether, glycocholateand deoxycholate, or oily solutions for administration in the form ofnasal drops, or as a gel to be applied intranasally. Formulations forparenteral administration may also include glycocholate for buccaladministration, methoxysalicylate for rectal administration, or cutricacid for vaginal administration. Suppositories for rectal administrationmay also be prepared by mixing the polymeric thyroid hormone analogsalone or in combination with nerve growth factors or other neurogenesisfactors, inducer or agonist with a non-irritating excipient such ascocoa butter or other compositions which are solid at room temperatureand liquid at body temperatures.

Formulations for topical administration to the skin surface may beprepared by dispersing the polymeric thyroid hormone analogs alone or incombination with nerve growth factors or other neurogenesis factors,inducer or agonist with a dermatologically acceptable carrier such as alotion, cream, ointment or soap. Particularly useful are carrierscapable of forming a film or layer over the skin to localize applicationand inhibit removal. For topical administration to internal tissuesurfaces, the agent may be dispersed in a liquid tissue adhesive orother substance known to enhance adsorption to a tissue surface. Forexample, hydroxypropylcellulose or fibrinogen/thrombin solutions may beused to advantage. Alternatively, tissue-coating solutions, such aspectin-containing formulations may be used.

Alternatively, the agents described herein may be administered orally.Oral administration of proteins as therapeutics generally is notpracticed, as most proteins are readily degraded by digestive enzymesand acids in the mammalian digestive system before they can be absorbedinto the bloodstream. However, the polymeric thyroid hormone analogsalone or in combination with nerve growth factors or other neurogenesisfactors described herein typically are acid stable andprotease-resistant (see, for example, U.S. Pat. No. 4,968,590). Inaddition, OP-1, has been identified in mammary gland extract, colostrumand 57-day milk. Moreover, the OP-1 purified from mammary gland extractis morphogenically-active and is also detected in the bloodstream.Maternal administration, via ingested milk, may be a natural deliveryroute of TGF-β superfamily proteins. Letterio, et al., Science 264:1936-1938 (1994), report that TGF-β is present in murine milk, and thatradiolabelled TGF-β is absorbed by gastrointestinal mucosa of sucklingjuveniles. Labeled, ingested TGF-β appears rapidly in intact form in thejuveniles' body tissues, including lung, heart and liver. Finally,soluble form polymeric thyroid hormone analogs alone or in combinationwith nerve growth factors or other neurogenesis factors, e.g., maturepolymeric thyroid hormone analogs alone or in combination with nervegrowth factors or other neurogenesis factors with or withoutanti-oxidant or anti-inflammatory agents. These findings, as well asthose disclosed in the examples below, indicate that oral and parenteraladministration are viable means for administering TGF-β superfamilyproteins, including the polymeric thyroid analog alone or in combinationwith nerve growth factor or other neurogenesis factors, to anindividual. In addition, while the mature forms of certain polymericthyroid analog alone or in combination with nerve growth factor or otherneurogenesis factors described herein typically are sparingly soluble,the polymeric thyroid analog alone or in combination with nerve growthfactor or other neurogenesis factors form found in milk (and mammarygland extract and colostrum) is readily soluble, probably by associationof the mature, morphogenically-active form with part or all of the prodomain of the expressed, full length polypeptide sequence and/or byassociation with one or more milk components. Accordingly, the compoundsprovided herein may also be associated with molecules capable ofenhancing their solubility in vitro or in vivo.

Where the polymeric thyroid hormone analogs alone or in combination withnerve growth factors or other neurogenesis factors is intended for useas a therapeutic for disorders of the CNS, an additional problem must beaddressed: overcoming the blood-brain barrier, the brain capillary wallstructure that effectively screens out all but selected categories ofsubstances present in the blood, preventing their passage into thebrain. The blood-brain barrier can be bypassed effectively by directinfusion of the polymeric thyroid hormone analogs into the brain, or byintranasal administration or inhalation of formulations suitable foruptake and retrograde transport by olfactory neurons. Alternatively, thepolymeric thyroid hormone analogs can be modified to enhance itstransport across the blood-brain barrier. For example, truncated formsof the polymeric thyroid hormone analogs alone or in combination withnerve growth factors or other neurogenesis factors or a polymericthyroid hormone analog alone or in combination with nerve growth factoror other neurogenesis factors-stimulating agent may be most successful.Alternatively, the polymeric thyroid hormone analogs alone or incombination with nerve growth factors or other neurogenesis factors,inducers or agonists provided herein can be derivatized or conjugated toa lipophilic moiety or to a substance that is actively transportedacross the blood-brain barrier, using standard means known to thoseskilled in the art. See, for example, Pardridge, Endocrine Reviews 7:314-330 (1986) and U.S. Pat. No. 4,801,575.

The compounds provided herein may also be associated with moleculescapable of targeting the polymeric thyroid hormone analogs alone or incombination with nerve growth factors or other neurogenesis factors,inducer or agonist to the desired tissue. For example, an antibody,antibody fragment, or other binding protein that interacts specificallywith a surface molecule on cells of the desired tissue, may be used.Useful targeting molecules may be designed, for example, using thesingle chain binding site technology disclosed in U.S. Pat. No.5,091,513. Targeting molecules can be covalently or non-covalentlyassociated with the polymeric thyroid hormone analogs alone or incombination with nerve growth factors or other neurogenesis factors,inducer or agonist.

As will be appreciated by one of ordinary skill in the art, theformulated compositions contain therapeutically-effective amounts of thepolymeric thyroid hormone analogs alone or in combination with nervegrowth factors or other neurogenesis factors, inducers or agoniststhereof. That is, they contain an amount which provides appropriateconcentrations of the agent to the affected nervous system tissue for atime sufficient to stimulate a detectable restoration of impairedcentral or peripheral nervous system function, up to and including acomplete restoration thereof. As will be appreciated by those skilled inthe art, these concentrations will vary depending upon a number offactors, including the biological efficacy of the selected agent, thechemical characteristics (e.g., hydrophobicity) of the specific agent,the formulation thereof, including a mixture with one or moreexcipients, the administration route, and the treatment envisioned,including whether the active ingredient will be administered directlyinto a tissue site, or whether it will be administered systemically. Thepreferred dosage to be administered is also likely to depend onvariables such as the condition of the diseased or damaged tissues, andthe overall health status of the particular mammal. As a general matter,single, daily, biweekly or weekly dosages of 0.00001-1000 mg of apolymeric thyroid analog alone or in combination with nerve growthfactor or other neurogenesis factors are sufficient in the presence ofanti-oxidant and/or anti-inflammatory agents, with 0.0001-100 mg beingpreferable, and 0.001 to 10 mg being even more preferable.Alternatively, a single, daily, biweekly or weekly dosage of 0.01-1000.mu.g/kg body weight, more preferably 0.01-10 mg/kg body weight, may beadvantageously employed. The present effective dose can be administeredin a single dose or in a plurality (two or more) of installment doses,as desired or considered appropriate under the specific circumstances. Abolus injection or diffusable infusion formulation can be used. Ifdesired to facilitate repeated or frequent infusions, implantation of asemi-permanent stent (e.g., intravenous, intraperitoneal, intracisternalor intracapsular) may be advisable.

The polymeric thyroid hormone analogs alone or in combination with nervegrowth factors or other neurogenesis factors, inducers or agonists ofthe invention may, of course, be administered alone or in combinationwith other molecules known to be beneficial in the treatment of theconditions described herein. For example, various well-known growthfactors, hormones, enzymes, therapeutic compositions, antibiotics, orother bioactive agents can also be administered with the polymericthyroid hormone analogs alone or in combination with nerve growthfactors or other neurogenesis factors. Thus, various known growthfactors such as NGF, EGF, PDGF, IGF, FGF, TGF-α, and TGF-β, as well asenzymes, enzyme inhibitors, antioxidants, anti-inflammatory agents, freeradical scavenging agents, antibiotics and/orchemoattractant/chemotactic factors, can be included in the presentpolymeric thyroid hormone analogs alone or in combination with nervegrowth factors or other neurogenesis factors formulation.

Materials & Methods

Reagents: All reagents were chemical grade and purchased from SigmaChemical Co. (St. Louis, Mo.) or through VWR Scientific (Bridgeport,N.J.). Cortisone acetate, bovine serum albumin (BSA) and gelatinsolution (2% type B from bovine skin) were purchased from Sigma ChemicalCo. Fertilized chicken eggs were purchased from Charles RiverLaboratories, SPAFAS Avian Products & Services (North Franklin, Conn.).T4, 3,5,3′-triiodo-L-thyronine (T3), tetraiodothyroacetic acid (tetrac),T4-agarose, 6-N-propyl-2-thiouracil (PTU), RGD-containing peptides, andRGE-containing peptides were obtained from Sigma; PD 98059 fromCalbiochem; and CGP41251 was a gift from Novartis Pharma (Basel,Switzerland). Polyclonal anti-FGF2 and monoclonal anti-β-actin wereobtained from Santa Cruz Biotechnology and human recombinant FGF2 andVEGF from Invitrogen. Polyclonal antibody to phosphorylated ERK1/2 wasfrom New England Biolabs and goat anti-rabbit IgG from DAKO. Monoclonalantibodies to αVβ3 (SC73 12) and α-tubulin (E9) were purchased fromSanta Cruz Biotechnology (Santa Cruz, Calif.). Normal mouse IgG andHRP-conjugated goat anti-rabbit Ig were purchased from Dako Cytomation(Carpinteria, Calif.). Monoclonal antibodies to αVβ3 (LM609) and αVβ5(P1F6), as well as purified αVβ3, were purchased from Chemicon(Temecula, Calif.). L-[¹²⁵I]-T4 (specific activity, 1250 μCi/μg) wasobtained from Perkin Elmer Life Sciences (Boston, Mass.).

Chorioallantoic membrane (CAM) Model of Angiogenesis: In vivoNeovascularization was examined by methods described previously. 9-12Ten-day-old chick embryos were purchased from SPAFAS (Preston, Conn.)and incubated at 37° C. with 55% relative humidity. A hypodermic needlewas used to make a small hole in the shell concealing the air sac, and asecond hole was made on the broad side of the egg, directly over anavascular portion of the embryonic membrane that was identified bycandling. A false air sac was created beneath the second hole by theapplication of negative pressure at the first hole, causing the CAM toseparate from the shell. A window approximately 1.0 cm 2 was cut in theshell over the dropped CAM with a small-crafts grinding wheel (Dremel,division of Emerson Electric Co.), allowing direct access to theunderlying CAM. FGF2 (1 μg/mL) was used as a standard proangiogenicagent to induce new blood vessel branches on the CAM of 10-day-oldembryos. Sterile disks of No. 1 filter paper (Whatman International)were pretreated with 3 mg/mL cortisone acetate and 1 mmol/L PTU and airdried under sterile conditions. Thyroid hormone, hormone analogues, FGF2or control solvents, and inhibitors were then applied to the disks andthe disks allowed to dry. The disks were then suspended in PBS andplaced on growing CAMs. Filters treated with T4 or FGF2 were placed onthe first day of the 3-day incubation, with antibody to FGF2 added 30minutes later to selected samples as indicated. At 24 hours, the MAPKcascade inhibitor PD 98059 was also added to CAMs topically by means ofthe filter disks.

Microscopic Analysis of CAM Sections: After incubation at 37° C. with55% relative humidity for 3 days, the CAM tissue directly beneath eachfilter disk was resected from control and treated CAM samples. Tissueswere washed 3× with PBS, placed in 35-mm Petri dishes (Nalge Nunc), andexamined under an SV6 stereomicroscope (Zeiss) at ×50 magnification.Digital images of CAM sections exposed to filters were collected using a3-charge-coupled device color video camera system (Toshiba) and analyzedwith Image-Pro software (Media Cybernetics). The number of vessel branchpoints contained in a circular region equal to the area of each filterdisk were counted. One image was counted in each CAM preparation, andfindings from 8 to 10 CAM preparations were analyzed for each treatmentcondition (thyroid hormone or analogues, FGF2, FGF2 antibody, PD 98059).In addition, each experiment was performed 3 times. The resultingangiogenesis index is the mean ±SEM of new branch points in each set ofsamples.

FGF2 Assays: ECV304 endothelial cells were cultured in M199 mediumsupplemented with 10% fetal bovine serum. ECV304 cells (10⁶ cells) wereplated on 0.2% gel-coated 24-well plates in complete medium overnight,and the cells were then washed with serum-free medium and treated withT4 or T3 as indicated. After 72 hours, the supernatants were harvestedand assays for FGF performed without dilution using a commercial ELISAsystem (R&D Systems).

MAPK Activation: ECV304 endothelial cells were cultured in M199 mediumwith 0.25% hormone-depleted serum 13 for 2 days. Cells were then treatedwith T4 (10⁻⁷ mol/L) for 15 minutes to 6 hours. In additionalexperiments, cells were treated with T4 or FGF2 or with T4 in thepresence of PD 98059 or CGP41251. Nuclear fractions were pre-pared fromall samples by our method reported previously, the proteins separated bypolyacrylamide gel electrophoresis, and transferred to membranes forimmunoblotting with antibody to phosphorylated ERK 1/2. The appearanceof nuclear phosphorylated ERK1/2 signifies activation of these MAPKisoforms by T4.

Reverse Transcription-Polymerase Chain Reaction: Confluent ECV304 cellsin 10-cm plates were treated with T4 (10⁻⁷ mol/L) for 6 to 48 hours andtotal RNA extracted using guanidinium isothiocyanate (BiotecxLaboratories). RNA (1 μg) was subjected to reversetranscription-polymerase chain reaction (RT-PCR) using the Access RT-PCRsystem (Promega). Total RNA was reverse transcribed into cDNA at 48° C.for 45 minutes, then denatured at 94° C. for 2 minutes. Second-strandsynthesis and PCR amplification were performed for 40 cycles withdenaturation at 94° C. for 30 s, annealing at 60° C. for 60 s, andextension at 68° C. for 120 s, with final ex-tension for 7 minutes at68° C. after completion of all cycles. PCR primers for FGF2 were asfollows: FGF2 sense strand 5′-TGGTATGTGGCACTGAAACG-3′ (SEQ ID NO:1),antisense strand 5′ CTCAATGACCTGGCGAAGAC-3′ (SEQ ID NO:2); the length ofthe PCR product was 734 bp. Primers for GAPDH included the sense strand5′-AAGGTCATCCCTGAGCTGAACG-3′ (SEQ ID NO:3), and antisense strand5′-GGGTGTCGCTGTTGAAGTCAGA-3′ (SEQ ID NO:4); the length of the PCRproduct was 218 bp. The products of RT-PCR were separated byelectrophoresis on 1.5% agarose gels and visualized with ethidiumbromide. The target bands of the gel were quantified using LabImagesoftware (Kapelan), and the value for [FGF2/GAPDH]X10 calculated foreach time point.

Statistical Analysis: Statistical analysis was performed by 1-wayanalysis of variance (ANOVA) comparing experimental with respectivecontrol group and statistical significance was calculated based onP<0.05.

In vivo angiogenesis in Matrigel FGF₂ or Cancer cell lines implant inmice: In Vivo Murine Angiogenesis Model: The murine matrigel model willbe conducted according to previously described methods (Grant et al.,1991; Okada et al., 1995) and as implemented in our laboratory (Powel etal., 2000). Briefly, growth factor free matrigel (Becton Dickinson,Bedford Mass.) will be thawed overnight at 4° C. and placed on ice.Aliquots of matrigel will be placed into cold polypropylene tubes andFGF2, thyroid hormone analogs or cancer cells (1×10⁶ cells) will beadded to the matrigel. Matrigel with Saline, FGF2, thyroid hormoneanalogs or cancer cells will be subcutaneously injected into the ventralmidline of the mice. At day 14, the mice will be sacrificed and thesolidified gels will be resected and analyzed for presence of newvessels. Compounds A-D will be injected subcutaneously at differentdoses. Control and experimental gel implants will be placed in a microcentrifuge tube containing 0.5 ml of cell lysis solution (Sigma, St.Louis, Mo.) and crushed with a pestle. Subsequently, the tubes will beallowed to incubate overnight at 4° C. and centrifuged at 1,500×g for 15minutes on the following day. A 200 μl aliquot of cell lysate will beadded to 1.3 ml of Drabkin's reagent solution (Sigma, St. Louis, Mo.)for each sample. The solution will be analyzed on a spectrophotometer ata 540 nm. The absorption of light is proportional to the amount ofhemoglobin contained in the sample.

Tumor growth and metastasis—Chick Chorioallantoic Membrane (CAM) modelof tumor implant: The protocol is as previously described (Kim et al.,2001). Briefly, 1×10⁷ tumor cells will be placed on the surface of eachCAM (7 day old embryo) and incubated for one week. The resulting tumorswill be excised and cut into 50 mg fragments. These fragments will beplaced on additional 10 CAMs per group and treated topically thefollowing day with 25 μl of compounds (A-D) dissolved in PBS. Seven dayslater, tumors will then be excised from the egg and tumor weights willbe determined for each CAM. FIG. 8 is a diagrammatic sketch showing thesteps involved in the in vivo tumor growth model in the CAM.

The effects of TETRAC, TRIAC, and thyroid hormone antagonists on tumorgrowth rate, tumor angiogenesis, and tumor metastasis of cancer celllines can be determined.

Tumor growth and metastasis—Tumor Xenograft model in mice: The model isas described in our publications by Kerr et al., 2000; Van Waes et al.,2000; Ali et al., 2001; and Ali et al., 2001, each of which isincorporated herein by reference in its entirety). The anti-cancerefficacy for TETRAC, TRIAC, and other thyroid hormone antagonists atdifferent doses and against different tumor types can be determined andcompared.

Tumor growth and metastasis—Experimental Model of Metastasis: The modelis as described in our recent publications (Mousa, 2002; Amirkhosravi etal., 2003a and 2003b, each of which is incorporated by reference hereinin its entirety). Briefly, B16 murine malignant melanoma cells (ATCC,Rockville, Md.) and other cancer lines will be cultured in RPMI 1640(Invitrogen, Carlsbad, Calif.), supplemented with 10% fetal bovineserum, penicillin and streptomycin (Sigma, St. Louis, Mo.). Cells willbe cultured to 70% confluency and harvested with trypsin-EDTA (Sigma)and washed twice with phosphate buffered saline (PBS). Cells will bere-suspended in PBS at a concentration of either 2.0×10⁵ cells/ml forexperimental metastasis. Animals: C57/BL6 mice (Harlan, Indianapolis,Ind.) weighing 18-21 grams will be used for this study. All proceduresare in accordance with IACUC and institutional guidelines. Theanti-cancer efficacy for TETRAC, TRIAC, and other thyroid hormoneantagonists at different doses and against different tumor types can bedetermined and compared.

Effect of Thyroid Hormone Analogues on Angiogenesis.

T4 induced significant increase in angiogenesis index (fold increaseabove basal) in the CAM model. T3 at 0.001-1.0 μM or T4 at 0.1-1.0 μMachieved maximal effect in producing 2-2.5 fold increase in angiogenesisindex as compared to 2-3 fold increase in angiogenesis index by 1 μg ofFGF2 (Table 1 and FIG. 1 a and 1 b). The effect of T4 in promotingangiogenesis (2-2.5 fold increase in angiogenesis index) was achieved inthe presence or absence of PTU, which inhibit T4 to T3 conversion. T3itself at 91-100 nM)-induced potent pro-angiogenic effect in the CAMmodel. T4 agarose produced similar pro-angiogenesis effect to thatachieved by T4. The pro-angiogenic effect of either T4 or T4-agarose was100% blocked by TETRAC or TRIAC.

Enhancement of pro-angiogenic activity of FGF2 by sub-maximalconcentrations of T₄.

The combination of T4 and FGF2 at sub-maximal concentrations resulted inan additive increase in the angiogenesis index up to the same level likethe maximal pro-angiogenesis effect of either FGF2 or T4 (FIG. 2).

Effects of MAPK cascade inhibitors on the pro-angiogenic actions of T₄and FGf2 n the CAM model. The pro-angiogenesis effect of either T4 orFGF2 was totally blocked by PD 98059 at 0.8-8 μg (FIG. 3).

Effects of specific integrin αVβ3 antagonists on the pro-angiogenicactions of T₄ and FGf2 n the CAM model. The pro-angiogenesis effect ofeither T4 or FGF2 was totally blocked by the specific monoclonalantibody LM609 at 10 μg (FIGS. 4 a and 4 b).

The CAM assay has been used to validate angiogenic activity of a varietyof growth factors and other promoters or inhibitors of angiogenesis. Inthe present studies, T₄ in physiological concentrations was shown to bepro-angiogenic, with comparable activity to that of FGF2. The presenceof PTU did not reduce the effect of T₄, indicating that de-iodination ofT₄ to generate T₃ was not a prerequisite in this model. Because theappearance of new blood vessel growth in this model requires severaldays, we assumed that the effect of thyroid hormone was totallydependent upon the interaction of the nuclear receptor for thyroidhormone (TR). Actions of iodothyronines that require intranuclearcomplexing of TR with its natural ligand, T₃, are by definition,genomic, and culminate in gene expression. On the other hand, thepreferential response of this model system to T₄-rather than T₃, thenatural ligand of TR raised the possibility that angiogenesis might beinitiated non-gnomically at the plasma membrane by T₄ and culminate ineffects that require gene transcription. Non-genomic actions of T₄ havebeen widely described, are usually initiated at the plasma membrane andmay be mediated by signal transduction pathways. They do not requireintranuclear ligand binding of iodothyronine and TR, but may interfacewith or modulate gene transcription. Non-genomic actions of steroidshave also been well-described and are known to interface with genomicactions of steroids or of other compounds. Experiments carried out withT₄ and tetrac or with agarose-T₄ indicated that the pro-angiogeniceffect of T₄ indeed very likely was initiated at the plasma membrane. Wehave shown elsewhere that tetrac blocks membrane-initiated effects ofT₄, but does not, itself, activate signal transduction. Thus, it is aprobe for non-genomic actions of thyroid hormone. Agarose-T₄ is thoughtnot to gain entry to the cell interior and has been used by us andothers to examine models for possible cell surface-initiated actions ofthe hormone.

These results suggest that another consequence of activation of MAPK bythyroid hormone is new blood vessel growth. The latter is initiatednongenomically, but of course requires a consequent complex genetranscription program.

The ambient concentrations of thyroid hormone are relatively stable. TheCAM model, at the time we tested it, was thyroprival and thus may beregarded as a system, which does not reproduce the intact organism. Wepropose that circulating levels of T₄ serve, with a variety of otherregulators, to modulate the sensitivity of vessels to endogenousangiogenic factors, such as VEGF and FGF2.

Three-Dimensional Angiogenesis Assay

In Vitro Three-Dimensional Sprout Angiogenesis of Human DermalMicro-Vascular Endothelial Cells (HDMEC) Cultured on Micro-Carrier BeadsCoated with Fibrin: Confluent HDMEC (passages 5-10) were mixed withgelatin-coated Cytodex-3 beads with a ratio of 40 cells per bead. Cellsand beads (150-200 beads per well for 24-well plate) were suspended with5 ml EBM+15% normal human serum, mixed gently every hour for first 4hours, then left to culture in a CO₂ incubator overnight. The next day,10 ml of fresh EBM+5% HS were added, and the mixture was cultured foranother 3 hours. Before experiments, the culture of EC-beads waschecked; then 500 ul of PBS was added to a well of 24-well plate, and100 ul of the EC-bead culture solution was added to the PBS. The numberof beads was counted, and the concentration of EC/beads was calculated.

A fibrinogen solution (1 mg/ml) in EBM medium with or withoutangiogenesis factors or testing factors was prepared. For positivecontrol, 50 ng/ml VEGF+25 ng/ml FGF2 was used. EC-beads were washed withEBM medium twice, and EC-beads were added to fibrinogen solution. Theexperiment was done in triplicate for each condition. The EC-beads weremixed gently in fibrinogen solution, and 2.5 ul human thrombin (0.05U/ul) was added in 1 ml fibrinogen solution; 300 ul was immediatelytransferred to each well of a 24-well plate. The fibrinogen solutionpolymerizes in 5-10 minutes; after 20 minutes, we added EBM+20% normalhuman serum+10 ug/ml aprotinin. The plate was incubated in a CO₂incubator. It takes about 24-48 hours for HDMEC to invade fibrin gel andform tubes.

A micro-carrier in vitro angiogenesis assay previously designed toinvestigate bovine pulmonary artery endothelial cell angiogenic behaviorin bovine fibrin gels [Nehls and Drenckhaln, 1995a, b] was modified forthe study of human microvascular endothelial cell angiogenesis inthree-dimensional ECM environments (FIGS. 1 and 2). Briefly, humanfibrinogen, isolated as previously described [Feng et al, 1999], wasdissolved in M199 medium at a concentration of 1 mg/ml (pH 7.4) andsterilized by filtering through a 0.22 micron filter. An isotonic 1.5mg/ml collagen solution was prepared by mixing sterile Vitrogen 100 in5×M199 medium and distilled water. The pH was adjusted to 7.4 by 1NNaOH. In certain experiments, growth factors and ECM proteins (such asVEGF, bFGF, PDGF-BB, serum, gelatin, and fibronectin) were added to thefibrinogen or collagen solutions. About 500 EC-beads were then added tothe 1 mg/ml fibrinogen or 1.5 mg/ml collagen solutions. Subsequently,EC-beads-collagen or EC-beads-fibrinogen suspension (500 EC-beads/ml)was plated onto 24-well plates at 300 ul/well. EC-bead-collagen cultureswere incubated at 37° C. to form gel. The gelling of EC-bead-fibrincultures occurred in less than 5 minutes at room temperature after theaddition of thrombin to a final concentration of 0.5 U/ml. Aftergelation, 1 ml of fresh assay medium (EBM supplemented with 20% normalhuman serum for HDMEC or EBM supplemented with 10% fetal bovine serumwas added to each well. The angiogenic response was monitored visuallyand recorded by video image capture. Specifically, capillary sproutformation was observed and recorded with a Nikon Diaphot-TMD invertedmicroscope (Nikon Inc.; Melville, N.Y.), equipped with an incubatorhousing with a Nikon NP-2 thermostat and Sheldon #2004 carbon dioxideflow mixer. The microscope was directly interfaced to a video systemconsisting of a Dage-MTI CCD-72S video camera and Sony 12″ PVM-122 videomonitor linked to a Macintosh G3 computer. The images were captured atvarious magnifications using Adobe Photoshop. The effect of angiogenicfactors on sprout angiogenesis was quantified visually by determiningthe number and percent of EC-beads with capillary sprouts. One hundredbeads (five to six random low power fields) in each of triplicate wellswere counted for each experimental condition. All experiments wererepeated at least three times.

Cell Culture.

The African green monkey fibroblast cell line, CV-1 (ATCC, Manassas,Va.), which lacks the nuclear receptor for thyroid hormone, was platedat 5000 cells/cm² and maintained in DMEM, supplemented with 10% (v/v)heat-inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2mM L-glutamine. All culture reagents were purchased from InvitrogenCorporation (Carlsbad, Calif.). Cultures were maintained in a 37° C.humidified chamber with 5% CO₂. The medium was changed every three daysand the cell lines were passaged at 80% confluency. For experimentaltreatment, cells were plated in 10-cm cell culture dishes (CorningIncorporated, Corning, N.Y.) and allowed to grow for 24 h in 10%FBS-containing medium. The cells were then rinsed twice with phosphatebuffered saline (PBS) and fed with serum-free DMEM supplemented withpenicillin, streptomycin, and HEPES. After 48 h incubation in serum-freemedia, the cells were treated with a vehicle control (finalconcentration of 0.004 N KOH with 0.4% polyethyleneglycol [v/v]) or T4(10⁻⁷ M final concentration) for 30 min; media were then collected andfree T4 levels were determined by enzyme immunoassays. Culturesincubated with 10⁻⁷ M total T4 have 10⁻⁹ to 10⁻¹⁰ M free T4. Followingtreatment, the cells were harvested and the nuclear proteins prepared aspreviously described.

Transient Transfections with siRNA.

CV-1 cells were plated in 10-cm dishes (150,000 cells/dish) andincubated for 24 h in DMEM supplemented with 10% FBS. The cells wererinsed in OPTI-MEM (Ambion, Austin, Tex.) and transfected with siRNA(100 nM final concentration) to αV, β3, or αV and β3 together usingsiPORT (Ambion) according to manufacturer's directions. Additional setsof CV-1 cells were transfected with a scrambled siRNA, to serve as anegative control. Four hours post-transfection, 7 ml of 10%FBS-containing media was added to the dishes and the cultures wereallowed to incubate overnight. The cells were then rinsed with PBS andplaced in serum-free DMEM for 48 h before treatment with T4.

RNA Isolation and RT-PCR.

Total RNA was extracted from cell cultures 72 h post-transfection usingthe RNeasy kit from Qiagen (Valencia, Calif.) as per manufacturer'sinstructions. Two hundred nanograms of total RNA was reverse-transcribedusing the Access RT-PCR system (Promega, Madison, Wis.) according tomanufacturer's directions. Primers were based on publishedspecies-specific sequences: αV (accession number NM-002210)F-5′-TGGGATTGTGGAAGGAG and R-5′-AAATCCCTGTCCATCAGCAT (319 bp product),β3 (NM000212) F-5′-GTGTGAGTGCTCAGAGGAG and R-5′-CTGACTCAATCTCGTCACGG (515 bp product), and GAPDH (AF261085) F-5′-GTCAGTGGTGGACCTGACCT andR-5′-TGAGCTTGACMGTGGTCG (212 bp product). RT-PCR was performed in theFlexigene thermal cycler eom TECHNE (Burlington, N.J.). After a 2 minincubation at 95° C., 25 cycles of the following steps were performed:denaturation at 94° C. for 1 min, annealing at 57° C. for 1 min, andextension for 1 min at 68° C. for 25 cycles. The PCR products werevisualized on a 1.8% (w/v) agarose gel stained with ethidium bromide.

Western Blotting.

Aliquots of nuclear proteins (10 μg/lane) were mixed with Laemmli samplebuffer and separated by SDS-PAGE (10% resolving gel) and thentransferred to nitrocellulose membranes. After blocking with 5% non-fatmilk in Tris-buffered saline containing 1% Tween-20 (TBST) for 30 min,the membranes were incubated with a 1:1000 dilution of a monoclonalantibody to phosphorylated p44/42 MAP kinase (Cell Signaling Technology,Beverly, Mass.) in TBST with 5% milk overnight at 4° C. Following3×10-min washes in TBST, the membranes were incubated withHRP-conjugated goat anti-rabbit Ig (1:1000 dilution) from DakoCytomation(Carpinteria, Calif.) in TBST with 5% milk for 1 h at room temperature.The membranes were washed 3×5 min in TBST and immunoreactive proteinswere detected by chemiluminescence (ECL, Amersham). Band intensity wasdetermined using the VersaDoc 5000 Imaging system (Bio-Rad, Hercules,Calif.).

Radioligand Binding Assay.

Two μg of purified αVβ3 was mixed with indicated concentrations of testcompounds and allowed to incubate for 30 min at room temperature.[¹²⁵I]-T4 (2 μCi) was then added and the mixture was allowed to incubatean additional 30 min at 20° C. The samples were mixed with sample buffer(50% glycerol, 0.1M Tris-HCl, pH 6.8, and bromophenol blue) and runouton a 5% basic-native gel for 24 h at 45 mA in the cold. The apparatuswas disassembled and the gels were placed on filter paper, wrapped inplastic wrap, and exposed to film. Band intensity was determined usingthe VersaDoc 5000 Imaging system.

Chick Chorioallantoic Membrane (CAM) Assay (αVβ3 Studies).

Ten-day-old chick embryos were purchased £tom SPAFAS (Preston, Conn.)and were incubated at 37° C. with 55% relative humidity. A hypodermicneedle was used to make a small hole in the blunt end of the egg and asecond hole was made on the broad side of the egg, directly over anavascular portion of the embryonic membrane. Mild suction was applied tothe first hole to displace the air sac and drop the CAM away from theshell. Using a Dremel model craft drill (Dremel, Racine, Wis.), aapprox. 1.0 cm² window was cut in the shell over the false air sac,allowing access to the CAM. Sterile disks of No. 1 filter paper(Whatman, Clifton, N.J.) were pretreated with 3 mg/ml cortisone acetateand 1mnMmpropylthiouracil and air dried under sterile conditions.Thyroid hormone, control solvents, and the mAb LM609 were applied to thedisks and subsequently dried. The disks were then suspended in PBS andplaced on growing CAMS. After incubation for 3 days, the CAM beneath thefilter disk was resected and rinsed with PBS. Each membrane was placedin a 35 mm Petri dish and examined under an SV6 stereo-microscope at 50×magnification. Digital images were captured and analyzed with Image-Prosoftware (Mediacybemetics). The number of vessel branch points containedin a circular region equal to the filter disk were counted. One imagefrom each of 8-10 CAM preparations for each treatment condition wascounted, and in addition each experiment was performed 3 times.

The invention will be further illustrated in the following non-limitingexamples.

EXAMPLES Example 1 Effect of Thyroid Hormone on Angiogenesis

As seen in FIG. 1A and summarized in FIG. 1B, both L-T4 and L-T3enhanced angiogenesis in the CAM assay. T4, at a physiologic totalconcentration in the medium of 0.1 μmol/L, increased blood vessel branchformation by 2.5-fold (P<0.001). T3 (1 nmol/L) also stimulatedangiogenesis 2-fold. The possibility that T4 was only effective becauseof conversion of T4 to T3 by cellular 5′-monodeiodinase was ruled out bythe finding that the deiodinase inhibitor PTU had no inhibitory effecton angiogenesis produced by T4. PTU was applied to all filter disks usedin the CAM model. Thus, T4 and T3 promote new blood vessel branchformation in a CAM model that has been standardized previously for theassay of growth factors.

Example 2 Effects of T4-Agarose and Tetrac

We have shown previously that T4-agarose stimulates cellular signaltransduction pathways initiated at the plasma membrane in the samemanner as T4 and that the actions of T4 and T4-agarose are blocked by adeaminated iodothyronine analogue, tetrac, which is known to inhibitbinding of T4 to plasma membranes. In the CAM model, the addition oftetrac (0.1 μmol/L) inhibited the action of T4 (FIG. 2A), but tetracalone had no effect on angiogenesis (FIG. 2C). The action of T4-agarose,added at a hormone concentration of 0.1 μmol/L, was comparable to thatof T4 in the CAM model (FIG. 2B), and the effect of T4-agarose was alsoinhibited by the action of tetrac (FIG. 2B; summarized in 2C).

Example 3 Enhancement of Proangiogenic Activity of FGF2 by a SubmaximalConcentration of T4

Angiogenesis is a complex process that usually requires theparticipation of polypeptide growth factors. The CAM assay requires atleast 48 hours for vessel growth to be manifest; thus, the apparentplasma membrane effects of thyroid hormone in this model are likely toresult in a complex transcriptional response to the hormone. Therefore,we determined whether FGF2 was involved in the hormone response andwhether the hormone might potentiate the effect of subphysiologic levelsof this growth factor. T4 (0.05 μmol/L) and FGF2 (0.5 μg/mL)individually stimulated angiogenesis to a modest degree (FIG. 3). Theangiogenic effect of this submaximal concentration of FGF2 was enhancedby a subphysiologic concentration of T4 to the level caused by 1.0 μgFGF2 alone. Thus, the effects of submaximal hormone and growth factorconcentrations appear to be additive. To define more precisely the roleof FGF2 in thyroid hormone stimulation of angiogenesis, a polyclonalantibody to FGF2 was added to the filters treated with either FGF2 orT4, and angiogenesis was measured after 72 hours. FIG. 4 demonstratesthat the FGF2 antibody inhibited angiogenesis stimulated either by FGF2or by T4 in the absence of exogenous FGF2, suggesting that the T4 effectin the CAM assay was mediated by increased FGF2 expression. Control IgGantibody has no stimulatory or inhibitory effect in the CAM assay.

Example 4 Stimulation of FGF2 Release From Endothelial Cells by ThyroidHormone

Levels of FGF2 were measured in the media of ECV304 endothelial cellstreated with either T4 (0.1 μmol/L) or T3 (0.01 μmol/L) for 3 days. Asseen in the Table 2, T3 stimulated FGF2 concentration in the medium3.6-fold, whereas T4 caused a 1.4-fold increase. This finding indicatesthat thyroid hormone may enhance the angiogenic effect of FGF2, at leastin part, by increasing the concentration of growth factor available toendothelial cells.

TABLE 2 Effect of T4 and T3 on Release of FGF2 From ECV304 EndothelialCells Cell Treatment FGF2 (pg/mL/10⁶ cells) Control 27.7 ± 3.1 T3 (0.01μmol/L)  98.8 ± 0.5* T3 + PD 98059 (2 μmol/L) 28.4 ± 3.2 T3 + PD 98059(20 μmol/L) 21.7 ± 3.5 T4 (0.1 μmol/L)  39.2 ± 2.8† T4 + PD 98059 (2μmol/L) 26.5 ± 4.5 T4 + PD 98059 (20 μmol/L) 23.2 ± 4.8 *P < 0.001,comparing T3-treated samples with control samples by ANOVA; †P < 0.05,comparing T4-treated samples with control samples by ANOVA.

Example 5 Role of the ERK1/2 Signal Transduction Pathway in Stimulationof Angiogenesis by Thyroid Hormone and FGF2

A pathway by which T4 exerts a nongenomic effect on cells is the MAPKsignal transduction cascade, specifically that of ERK1/2 activation. Weknow that T4 enhances ERK1/2 activation by epidermal growth factor. Therole of the MAPK pathway in stimulation by thyroid hormone of FGF2expression was examined by the use of PD 98059 (2 to 20 μmol/L), aninhibitor of ERK1/2 activation by the tyrosine-threonine kinases MAPKkinase-1 (MEK1) and MEK. The data in the Table demonstrate that PD 98059effectively blocked the increase in FGF2 release from ECV304 endothelialcells treated with either T4 or T3. Parallel studies of ERK1/2inhibition were performed in CAM assays, and representative results areshown in FIG. 5. A combination of T3 and T4, each in physiologicconcentrations, caused a 2.4-fold increase in blood vessel branching, aneffect that was completely blocked by 3 μmol/L PD 98059 (FIG. 5A). FGF2stimulation of branch formation (2.2-fold) was also effectively blockedby this inhibitor of ERK1/2 activation (FIG. 5B). Thus, theproangiogenic effect of thyroid hormone begins at the plasma membraneand involves activation of the ERK1/2 pathway to promote FGF2 releasefrom endothelial cells. ERK1/2 activation is again required to transducethe FGF2 signal and cause new blood vessel formation.

Example 6 Action of Thyroid Hormone and FGF2 on MAPK Activation

Stimulation of phosphorylation and nuclear translocation of ERK1/2 MAPKswas studied in ECV304 cells treated with T4 (10⁻⁷ mol/L) for 15 minutesto 6 hours. The appearance of phosphorylated ERK1/2 in cell nucleioccurred within 15 minutes of T4 treatment, reached a maximal level at30 minutes, and was still apparent at 6 hours (FIG. 6A). This effect ofthe hormone was inhibited by PD 98059 (FIG. 6B), a result to be expectedbecause this compound blocks the phosphorylation of ERK1/2 by MAPKkinase. The traditional protein kinase C (PKC)-α, PKC-β, and PKC-γinhibitor CGP41251 also blocked the effect of the hormone on MAPKactivation in these cells, as we have seen with T4 in other cell lines.Thyroid hormone enhances the action of several cytokines and growthfactors, such as interferon-γ13 and epidermal growth factor. In ECV304cells, T4 enhanced the MAPK activation caused by FGF2 in a 15-minute coincubation (FIG. 6C). Applying observations made in ECV304 cells to theCAM model, we propose that the complex mechanism by which the hormoneinduces angiogenesis includes endothelial cell release of FGF2 andenhancement of the autocrine effect of released FGF2 on angiogenesis.

Example 7 RT-PCR in ECV304 Cells Treated with Thyroid Hormone

The final question addressed in studies of the mechanism of theproangiogenic action of T4 was whether the hormone may induce FGF2 geneexpression. Endothelial cells were treated with T4 (10⁻⁷ mol/L) for 6 to48 hours, and RT-PCR-based estimates of FGF2 and GAPDH RNA (inferredfrom cDNA measurements; FIG. 7) were performed. Increase in abundance ofFGF2 cDNA, corrected for GAPDH content, was apparent by 6 hours ofhormone treatment and was further enhanced by 48 hours.

Example 8A Retinal Neovascularization Model in Mice (Diabetic andNon-Diabetic)

To assess the pharmacologic activity of a test article on retinalneovascularization, Infant mice are exposed to a high oxygen environmentfor 7 days and allowed to recover, thereby stimulating the formation ofnew vessels on the retina. Test articles are evaluated to determine ifretinal neovascularization is suppressed. The retinas are examined withhematoxylin-eosin staining and with at least one stain, whichdemonstrates neovascularization (usually a Selectin stain). Other stains(such as PCNA, PAS, GFAP, markers of angiogenesis, etc.) can be used. Asummary of the model is below:

Animal Model

-   -   Infant mice (P7) and their dams are placed in a hyper-oxygenated        environment (70-80%) for 7 days.    -   On P12, the mice are removed from the oxygenated environment and        placed into a normal environment    -   Mice are allowed to recover for 5-7 days.    -   Mice are then sacrificed and the eyes collected.    -   Eyes are either frozen or fixed as appropriate    -   The eyes are stained with appropriate histochemical stains    -   The eyes are stained with appropriate immunohistochemical stains    -   Blood, serum, or other tissues can be collected    -   Eyes, with special reference to microvascular alterations, are        examined for any and all findings. Neovascular growth will be        semi quantitatively scored. Image analysis is also available.

Example 8B Thyroid Hormone and Diabetic Retinopathy

A protocol disclosed in J de la Cruz et al., J Pharmacol Exp Ther280:454-459, 1997, is used for the administration of Tetrac to rats thathave streptozotocin (STZ)-induced experimental diabetes and diabeticretinopathy. The endpoint is the inhibition by Tetrac of the appearanceof proliferative retinopathy (angiogenesis).

Example 9A Wound Healing and Hemostatic Treatment Using NovelPharmaceutical Polymeric Formulation of Thyroid Hormone and Analogs

The present invention also includes a novel wound healing and hemostatictreatment that include an immobilized thyroid hormone analog, preferablyT4 analogs, calcium chloride, and collagen. This novel formulationsignificantly controls both venous and arterial hemorrhage, reducesbleeding time, generates fibrin/platelet plug, releases platelet-derivedwound healing factors in a sustained manner in the presence of low levelcollagen, and safe. Development of such a wound healing and hemostaticdressing can be very valuable for short and long-term use in CombatCasualty Care. Pharmaceutical formulation of immobilized L-thyroxine(T4) and globular hexasaccharide in a hydrogel or dressing containingcollagen and calcium chloride can be optimized. This novel Wound healingand Hemostatic (WH formulation) treatment in hydrogel or dressing canalso include the addition of a microbicidal.

L-thyroxine conjugated to polymer or immobilized on agarose demonstratedpotent stimulation of angiogenesis through activation of an adhesioncell surface receptor (integrin αVβ3) leading to activation of anintracellular signaling event, which in turn leads to up-regulation ofvarious growth factor productions. Additionally, immobilized T4 inducedepithelial, fibroblast, and keratinocyte cell migration. Immobilized T4,but not T3 or other analogs, enhanced collagen-induced plateletaggregation and secretion, which would promote formation of thesubject's own platelet plug. Furthermore, immobilized T4 also promoteswhite blood cell migration, which could be critical for fightinginfection. Hence, immobilized T4 can help the body make more of acompound used to regenerate damaged blood vessels, and it also increasedthe amount of white blood cells that makes free radicals in the woundsite. Free radicals help clear potentially pathogenic bacteria from awound.

Thus, T4 or T4-agarose (10-100 nM), but not T3, DIPTA, or GC-1, iseffective in enhancing platelet aggregation and secretion(de-granulation). Accordingly, T4 (or analogs and polymeric conjugationsthereof, e.g., T4-agarose), in combination with 10 mM calcium chloride,and with or without collagen, is preferred for wound healing. See FIGS.23A-E.

Thromboelastography:

Thromboelastography (TEG) has been used in various hospital settingssince its development by Hartert in 1948. The principle of TEG is basedon the measurement of the physical viscoelastic characteristics of bloodclot. Clot formation was monitored at 37° C. in an oscillating plasticcylindrical cuvette (“cup”) and a coaxially suspended stationary piston(“pin”) with a 1 mm clearance between the surfaces, using a computerizedThrombelastograph (TEG Model 3000, Haemoscope, Skokie, Ill.). The cuposcillates in either direction every 4.5 seconds, with a 1 secondmid-cycle stationary period; resulting in a frequency of 0.1 Hz and amaximal shear rate of 0.1 per second. The pin is suspended by a torsionwire that acts as a torque transducer. With clot formation, fibrinfibrils physically link the cup to the pin and the rotation of the cupas affected by the viscoelasticity of the clot (Transmitted to the pin)is displayed on-line using an IBM-compatible personal computer andcustomized software (Haemoscope Corp., Skokie, Ill.). The torqueexperienced by the pin (relative to the cup's oscillation) is plotted asa function of time.

TEG assesses coagulation by measuring various parameters such as thetime latency for the initial initiation of the clot (R), the time toinitiation of a fixed clot firmness (k) of about 20 mm amplitude, thekinetic of clot development as measured by the angle (α), and themaximum amplitude of the clot (MA). The parameter A measures the widthof the tracing at any point of the MA. Amplitude A in mm is a functionof clot strength or elasticity. The amplitude on the TEG tracing is ameasure of the rigidity of the clot; the peak strength or the shearelastic modulus attained by the clot, G, is a function of clot rigidityand can be calculated from the maximal amplitude (MA) of the TEGtracing.

The following parameters were measured from the TEG tracing:

-   -   R, the reaction time (gelation time) represents the latent        period before the establishment of a 3-dimensional fibrin gel        network (with measurable rigidity of about 2 mm amplitude).    -   Maximum Amplitude (MA, in mm), is the peak rigidity manifested        by the clot.    -   Shear elastic modulus or clot strength (G, dynes/cm²) is defined        by: G=(5000A)/(100-A).

Blood clot firmness is an important parameter for in vivo thrombosis andhemostasis because the clot must stand the shear stress at the site ofvascular injury. TEG can assess the efficacy of differentpharmacological interventions on various factors (coagulationactivation, thrombin generation, fibrin formation, platelet activation,platelet-fibrin interaction, and fibrin polymerization) involved in clotformation and retraction. The effect of endotoxin (0.63 ug), Xa (0.25μM), thrombin (0.3 mU), and TF (25 ng) on the different clot parametersmeasured by computerized TEG in human whole blood is shown in Table 3.

Blood Sampling: Blood was drawn from consenting volunteers under aprotocol approved by the Human Investigations Committee of WilliamBeaumont Hospital. Using the two syringe method, samples were drawnthrough a 21 gauge butterfly needle and the initial 3 ml blood wasdiscarded. Whole blood (WB) was collected into siliconized Vacutainertubes (Becton Dickinson, Rutherford, N.J. containing 3.8% trisodiumcitrate such that a ratio of citrate whole blood of 1:9 (v/v) wasmaintained. TEG was performed within 3 hrs of blood collection. Calciumwas added back at 1-2.5 mM followed by the addition of the differentstimulus. Calcium chloride by itself at the concentration used showedonly a minimal effect on clot formation and clot strength.

Clot formation is initiated by thrombin-induced cleavage ofFibrinopeptide A from fibrinogen. The resultant fibrin monomersspontaneously polymerize to form fibril strands that undergo linearextension, branching, and lateral association leading to the formationof a three-dimensional network of fibrin fibers. A unique property ofnetwork structures is that they behave as rigid elastic solids, capableof resisting deforming shear stress. This resistance to deformation canbe measured by elastic modulus-an index of clot strength. Unlikeconventional coagulation tests (like the prothrombin time and partialthromboplastin time) that are based only on the time to the onset ofclot formation, TEG allows acquisition of quantitative informationallowing measurement of the maximal strength attained by clots. Via theGPIIb/IIIa receptor, platelets bind fibrin(ogen) and modulate theviscoelastic properties of clots. Our results demonstrated that clotstrength in TF-TEG is clearly a function of platelet concentration andplatelets augmented clot strength 8 fold under shear. Different plateletGPIIb/IIIa antagonists (class I versus class II) behaved with distinctefficacy in inhibiting platelet-fibrin mediated clot strength usingTF-TEG under shear.

Statistical Analysis

Data are expressed as mean ±SEM. Data were analyzed by either paired orgroup analysis using the Student t test or ANOVA when applicable;differences were considered significant at P<0.05.

TABLE 3 Effect of Calcium Chloride versus Tissue Factor on clot dynamicsin citrated human whole blood using TEG 25 ng TF + 2.25 mM Ca⁺² 10 mMCa⁺² TEG Parameters (Mean ± SEM) (Mean ± SEM) r (min) 29.7 ± 2.3 14.5 ±2.5* K (min)  5.8 ± 1.0 7.0 ± 0.7 α (angle) 45.0 ± 2.6 47.3 ± 2.7  MA(mm) 58.2 ± 1.7 56.5 ± 2.2  Data represent mean ± SEM, n = 4, *P < 0.01.

Platelet aggregation and de-granulation in whole blood using ImpedanceTechnique: The Model 560 Whole-Blood Aggregometer and the associatedAggro-Link Software from the Chrono-Log Corporation were used in thisstudy. Two electrodes are placed in diluted blood and an electricalimpulse is sent from one to the other. As the platelets aggregate aroundthe electrodes, the Chrono-Log measures the impedance of the electricalsignal in ohms of resistance.

Blood Sampling:

Whole blood was drawn daily from healthy donors between the ages of 17and 21 into 4.5 milliliter Vacutainer vials with 3.8% buffered sodiumcitrate (Becton Dickinson, Rutherford, N.J.). The blood was kept on arocker to extend the life of the platelets, and experiments were donewithin 5 hours of phlebotomy.

Procedure: For the control, 500 microliters of whole blood, 500microliters of 0.9% saline, and a magnetic stir bar were mixed into acuvette, and heated for five minutes to 37 degrees Celsius.Sub-threshold aggregation was induced with 5 microliters of 1-2 μg/mlCollagen, which the Aggregometer measured for 6-7 minutes. The effectsof T4, T4-agarose versus T3 and other thyroid hormone analogs oncollagen-induced aggregation and secretion were tested.Ingerman-Wojenski C, Smith J B, Silver M J. Evaluation of electricalaggregometry: comparison with optical aggregometry, secretion of ATP,and accumulation of radiolabeled platelets. J Lab Clin Med. 1983January; 101(1):44-52.

Cell Migration Assay.

Human granulocytes are isolated from shed blood by the method of Mousaet al. and cell migration assays carried out as previously described(Methods In Cell Science, 19 (3): 179-187, 1997, and Methods In CellScience 19 (3): 189-195, 1997). Briefly, a neuroprobe 96 well disposablechemotaxis chamber with an 8 μm pore size will be used. This chamberallow for quantitation of cellular migration towards a gradient ofchemokine, cytokine or extracellular matrix proteins. Cell suspension(45 μl of 2×10⁶) will be added to a polypropylene plate containing 5 μlof test agents such as flavanoids or thyroid hormone derivatives andincubated for 10 minutes at 22° C. IL8 (0.1-100 ng) with or withoutT3/T4 (33 μL) at 0.001-0.1 μM will be added to the lower wells of adisposable chemotaxis chamber, then assemble the chamber using thepre-framed filter. Add 25 μl of cell/test agent suspension to the upperfilter wells then incubate overnight (22 hours at 37° C., 5% CO2) in ahumidified cell culture incubator. After the overnight incubation,non-migrated cells and excess media will be gently removed using a 12channel pipette and a cell scraper. The filters will then washed twicein phosphate buffered saline (PBS) and fixed with 1% formaldehyde in PBSbuffer. Membranes of migrated cells will be permeated with Triton X-100(0.2%) then washed 2-3 times with PBS. The actin filaments of migratedcells will be stained with Rhodamine phalloidin (12.8 IU/ml) for 30minutes (22° C.). Rhodamine phalloidin will be made fresh weekly andreused for up to 3 days, when stored protected from light at 4° C.Chemotaxis will be quantitatively determined by fluorescence detectionusing a Cytofluor II micro-filter fluorimeter (530 excitation/590emission). All cell treatments and subsequent washings will be carriedout using a uniquely designed treatment/wash station (Methods In CellScience, 19 (3): 179-187, 1997). This technique will allow for accuratequantitation of cell migration and provide reproducible results withminimal inter and intra assay variability.

Cellular Migration Assays:

These assays were performed using a Neuroprobe 96 well disposablechemotaxis chamber with an 8 μm pore size. This chamber allowed forquantitation of cellular migration towards a gradient of eithervitronectin or osteopontin. Cultured cells were removed following astandardized method using EDTA/Trypsin (0.01%/0.025%). Followingremoval, the cells were washed twice and resuspended (2×10⁶/ml) in EBM(Endothelial cell basal media, Clonetics Inc.). Add either vitronectinor osteopontin (33 μl) at 0.0125-100 μg/ml to the lower wells of adisposable chemotaxis chamber, and then assemble using the preframedfilter. The cell suspension (45 μl) was added to a polypropylene platecontaining 5 μl of test agent at different concentrations and incubatedfor 10 minutes at 22° C. Add 25 μl of cell/test agent suspension to theupper filter wells then incubate overnight (22 hours at 37° C.) in ahumidified cell culture incubator. After the overnight incubation,non-migrated cells and excess media were gently removed using a 12channel pipette and a cell scraper. The filters were then washed twicein PBS (no Ca⁺² or Mg⁺²) and fixed with 1% formaldehyde. Membranes ofmigrated cells were permeated with Triton X-100 (0.2%) then washed 2-3times with PBS. The actin filaments of migrated cells were stained withrhodamine phalloidin (12.8 IU/ml) for 30 minutes (22° C.). Rhodaminephalloidin was made fresh weekly and reused for up to 3 days, whenstored protected from light at 4° C. Chemotaxis was quantitativelydetermined by fluorescence detection using a Cytofluor II (530excitation/590 emission). All cell treatment and subsequent washingswere carried out using a uniquely designed treatment/wash station. Thisstation consisted of six individual reagent units each with a 30 mlvolume capacity. Individual units were filled with one of the followingreagents: PBS, formaldehyde, Triton X-100, or rhodamine-phalloidin.Using this technique, filters were gently dipped into the appropriatesolution, thus minimizing migrated cell loss. This technique allowed formaximum quantitation of cell migration and provided reproducible resultswith minimal inter and intra assay variability (1, 2).

Migration toward the extracellular Matrix Protein Vitronectin Treatments(Fluorescence Units) + SD Mean EC Migration A. Non-Specific Migration 270 ± 20 No Matrix in LC B. Vitronectin (25 ug) in LC 6,116 ± 185 C. T3(0.1 uM) UC/ 22,016 ± 385  Vitronectin (25 ug) in LC D. T4 (0.1 uM) UC/13,083 ± 276  Vitronectin (25 ug) in LC C + XT199 (10 uM) 4,550 ± 225D + XT199 (10 uM) 3,890 ± 420 C + PD (0.8 ug) 7,555 ± 320 D + PD (0.8ug) 6,965 ± 390 LC = Lower Chamber, UC = Upper chamber Similar data wereobtained with other potent and specific avb3 antagonists such as LM609and SM256

Example 9B In Vitro Human Epithelial and Fibroblast Wound Healing

The in vitro 2-dimensional wound healing method is as described inMohamed S, Nadijcka D, Hanson, V. Wound healing properties of cimetidinein vitro. Drug Intell Clin Pharm 20: 973-975; 1986, incorporated hereinby reference in its entirety. Additionally, a 3-dimensional woundhealing method already established in our Laboratory will be utilized inthis study (see below). Data show potent stimulation of wound healing bythyroid hormone.

In Vitro 3D Wound Healing Assay of Human Dermal Fibroblast Cells:

Step 1: Prepare Contracted Collagen Gels:

-   -   1) Coat 24-well plate with 350 ul 2% BSA at RT for 2 hr,    -   2) 80% confluent NHDF (normal human dermal fibroblast cells,        Passage 5-9) are trypsinized and neutralized with growth medium,        centrifuge and wash once with PBS    -   3) Prepare collagen-cell mixture, mix gently and always on ice:

Stock solution Final Concentration 5 × DMEC 1 × DMEM 3 mg/ml vitrogen 2mg/ml ddH2O optimal NHDF 2 × 10~5 cells/ml FBS 1%

-   -   4) Aspire 2% BSA from 24 well plate, add collagen-cell mixture        350 ul/well, and incubate the plate in 37° C. CO₂ incubator.    -   5) After 1 hr, add DMEM+5% FBS medium 0.5 ml/well, use a 10 ul        tip Detach the collagen gel from the edge of each well, then        incubate for 2 days. The fibroblast cells will contract the        collagen gel

Step 2: Prepare 3D Fibrin Wound Clot and Embed Wounded Collagen Culture

-   -   1) Prepare fibrinogen solution (1 mg/ml) with or without testing        regents. 350 ul fibrinogen solution for each well in eppendorf        tube.

Stock solution Final Concentration 5 × DMEC 1 × xDMEM Fibrinogen 1 mg/mlddH2O optimal testing regents optimal concentration FBS 1% or 5%

-   -   2) Cut each contracted collagen gel from middle with scissors.        Wash the gel with PBS and transfer the gel to the center of each        well of 24 well plate    -   3) Add 1.5 ul of human thrombin (0.25 U/ul) to each tube, mix        well and then add the solution around the collagen gel, the        solution will polymerize in 10 mins.

After 20 mins, add DMEM+1% (or 5%) FBS with or without testing agent,450 ul/well and incubate the plate in 37° C. CO₂ incubator for up to 5days. Take pictures on each day.

In Vivo Wound Healing in Diabetic Rats:

Using an acute incision wound model in diabetic rats, the effects ofthyroid hormone analogs and its conjugated forms are tested. The rate ofwound closure, breaking strength analyses and histology are performedperiodically on days 3-21.

Methods:

Animals (Mice and Rats) in the study are given two small puncturewounds—WH is applied to one of the wounds, and the other was coveredwith saline solution as a control. Otherwise, the wounds are left toheal naturally.

The animals are euthanised five days after they are wounded. A smallarea of skin—1 to 1.5 millimetres—is excised from the edges of thetreated and untreated wounds.

Wound closure and time to wound closure is determined. Additionally, thelevels of tenascin, a protein that helps build connective tissue, in thegranulation tissue of the wounds is determined. The quality of thegranulation tissue (i.e. rough, pinkish tissue that normally forms as awound heals, new capillaries and connective tissue) is also determined.

Materials and Methods

Chronic granulating wounds are prepared by methods well known in theart. Male Sprague Dawley rats weighing 300 to 350 grams are acclimatizedfor a week in our facility prior to use. Under intraperitoneal Nembutalanesthesia (35 mg/kg), the rat dorsum is shaved and depilated. Animalsare individually caged and given food and water ad libitum. Allexperiments were conducted in accordance with the Animal Care and UseCommittee guidelines of the Department of Veterans Affairs MedicalCenter, Albany, N.Y.

Histological characterization of this wound with comparison to a humanchronic granulating wound had previously been performed. Sixty four ratsare then divided into eight treatment groups (n=8/group). Animals aretreated with topical application of vehicle (vehicle controls) on days5, 9, 12, 15, and 18. The vehicle control can be either agarose(Group 1) or the polymeric form (Group 2) that will be used inconjugation of L-thyroxine. Wounds treated with T4-agarose (Groups 3-5)or T4-polymer (Groups 6-8) at 1, 10, 100 μg/cm² in the presence of 10 μgglobular hexasaccharide, 10 μg collagen, and 10 mM calcium chloride tobe applied topically on days 5, 9, 12, 15, and 18. All wounds are leftexposed. Every 48 hours the outlines of the wounds can be traced ontoacetate sheets, and area calculations can be performed usingcomputerized digital planimetry.

Three full-thickness, transverse strips of granulation tissue are thenharvested from the cephalad, middle, and caudal ends of the wounds onday 19 and fixed in 10-percent buffered formalin. Transverse sections (5μm) are taken from each specimen and stained with hematoxylin and eosin.The thickness of the granulation tissue can be estimated with an ocularmicrometer at low power. High-powered fields are examined immediatelybelow the superficial inflammatory layer of the granulation tissue. Fromeach strip of granulation tissue five adjacent high-powered fields canbe photographed and coded. Enlarged prints of these exposures are thenused for histometric analysis in a blinded fashion. Fibroblasts, “round”cells (macrophages, lymphocytes, and neutrophils), and capillaries arecounted. In addition the cellularity of each section is graded forcellularity on a scale of 1 (reduced cell counts) to 5 (highlycellular).

Statistical Analysis:

Serial area measurements were plotted against time. For each animal'sdata a Gompertz equation will be fitted (typical r 2=0.85). Using thiscurve the wound half-life can be estimated. Comparison between groups isperformed using life table analysis and the Wilcoxon rank test. Thesestatistical analyses are performed using the SAS (SAS/STAT Guide forPersonal Computers, Version 6 Edition, Cary, N.C., 1987, p 1028) andBMDP (BMDP Statistical Software Manual, Los Angeles, BMDP StatisticalSoftware, Inc. 1988) packages on a personal computer.

Cell counts for the different treatment groups are pooled and analyzedusing a one-way analysis of variance. Post-hoc analyses of differencesbetween groups can be carried out using Tukey's test (all pairwisemultiple-comparison test) with p<0.05 considered significant. Sigma Statstatistical software (Jandel Scientific, Corte Madera, Calif.) will beused for data analysis.

Example 10 Rodent Model of Myocardial Infarction

The coronary artery ligation model of myocardial infarction is used toinvestigate cardiac function in rats. The rat is initially anesthetizedwith xylazine and ketamine, and after appropriate anesthesia isobtained, the trachea is intubated and positive pressure ventilation isinitiated. The animal is placed supine with its extremities looselytaped and a median sternotomy is performed. The heart is gentlyexteriorized and a 6-O suture is firmly tied around the left anteriordescending coronary artery. The heart is rapidly replaced in the chestand the thoracotomy incision is closed with a 3-O purse string suturefollowed by skin closure with interrupted sutures or surgical clips.Animals are placed on a temperature regulated heating pad and closelyobserved during recovery. Supplemental oxygen and cardiopulmonaryresuscitation are administered if necessary. After recovery, the rat isreturned to the animal care facility. Such coronary artery ligation inthe rat produces large anterior wall myocardial infarctions. The 48 hr.mortality for this procedure can be as high as 50%, and there isvariability in the size of the infarct produced by this procedure. Basedon these considerations, and prior experience, to obtain 16-20 rats withlarge infarcts so that the two models of thyroid hormone deliverydiscussed below can be compared, approximately 400 rats are required.

These experiments are designed to show that systemic administration ofthyroid hormone either before or after coronary artery ligation leads tobeneficial effects in intact animals, including the extent ofhemodynamic abnormalities assessed by echocardiography and hemodynamicmeasurements, and reduction of infarct size. Outcome measurements areproposed at three weeks post-infarction. Although some rats may have noinfarction, or only a small infarction is produced, these rats can beidentified by normal echocardiograms and normal hemodynamics (LVend-diastolic pressure<8 mm Hg).

Thyroid Hormone Delivery

There are two delivery approaches. In the first, thyroid hormone isdirectly injected into the peri-infarct myocardium. As the demarcationbetween normal and ischemic myocardium is easily identified during theacute open chest occlusion, this approach provides sufficient deliveryof hormone to detect angiogenic effects.

Although the first model is useful in patients undergoing coronaryartery bypass surgery, and constitutes proof of principle that one localinjection induces angiogenesis, a broader approach using a second modelcan also be used. In the second model, a catheter retrograde is placedinto the left ventricle via a carotid artery in the anesthetized ratprior to inducing myocardial infarction. Alternatively, a direct needlepuncture of the aorta, just above the aortic valve, is performed. Theintracoronary injection of the thyroid hormone is then simulated byabruptly occluding the aorta above the origin of the coronary vesselsfor several seconds, thereby producing isovolumic contractions. Thyroidhormone is then injected into the left ventricle or aorta immediatelyafter aortic constriction. The resulting isovolumic contractions propelblood down the coronary vessels perfusing the entire myocardium withthyroid hormone. This procedure can be done as many times as necessaryto achieve effectiveness. The number of injections depends on the dosesused and the formation of new blood vessels.

Echocardiography:

A method for obtaining 2-D and M-mode echocardiograms in unanesthetizedrats has been developed. Left ventricular dimensions, function, wallthickness and wall motion can be reproducibly and reliably measured. Themeasurement are carried out in a blinded fashion to eliminate bias withrespect to thyroid hormone administration.

Hemodynamics:

Hemodynamic measurements are used to determine the degree of leftventricular impairment. Rats are anesthetized with isoflurane. Throughan incision along the right anterior neck, the right carotid artery andthe right jugular vein are isolated and cannulated with a pressuretransducing catheter (Millar, SPR-612, 1.2 Fr). The followingmeasurements are then made: heart rate, systolic and diastolic BP, meanarterial pressure, left ventricular systolic and end-diastolic pressure,and + and −dP/dt. Of particular utility are measurements of leftventricular end-diastolic pressure, progressive elevation of whichcorrelates with the degree of myocardial damage.

Infarct Size:

Rats are sacrificed for measurement of infarct size using TTCmethodology.

Morphometry

Microvessel density [microvessels/mm²] will be measured in the infarctarea, peri-infarct area, and in the spared myocardium opposing theinfarction, usually the posterior wall. From each rat, 7-10 microscopichigh power fields [×400] with transversely sectioned myocytes will bedigitally recorded using Image Analysis software. Microvessels will becounted by a blinded investigator. The microcirculation will be definedas vessels beyond third order arterioles with a diameter of 150micrometers or less, supplying tissue between arterioles and venules. Tocorrect for differences in left ventricular hypertrophy, microvesseldensity will be divided by LV weight corrected for body weight.Myocardium from sham operated rats will serves as controls.

Example 11 Effects of the αVβ3 antagonists on the pro-angiogenesiseffect of T4 or FGF2

The αVβ3 inhibitor LM609 totally inhibited both FGF2 or T4-inducedpro-angiogenic effects in the CAM model at 10 micrograms (FIG. 16).

Example 12 Inhibition of Cancer-Related New Blood Vessel Growth

A protocol disclosed in J. Bennett, Proc Natl Acad Sci USA 99:2211-2215,2002, is used for the administration of tetraiodothyroacetic (Tetrac) toSCID mice that have received implants of human breast cancer cells(MCF-7). Tetrac is provided in drinking water to raise the circulatinglevel of the hormone analog in the mouse model to 10⁻⁶ M. The endpointis the inhibitory action of tetrac on angiogenesis about the implantedtumors.

Example 13 Pro-Angiogenesis Promoting Effect of Thyroid Hormone andAnalogs Thereof at Subthreshold Levels of VEGF and FGF2 in an In VitroThree-Dimensional Micro-Vascular Endothelial Sprouting Model

Either T₃, T₄, T₄-agarose, or fibroblast growth factor 2 (FGF2) plusvascular endothelial growth factor (VEGF) produced a comparablepro-angiogenesis effect in the in vitro three-dimensional micro-vascularendothelial sprouting model. The pro-angiogenesis effect of the thyroidhormone analogs were blocked by PD 98059, an inhibitor of themitogen-activated protein kinase (MAPK; ERK1/2) signal transductioncascade. Additionally, a specific αVβ3 integrin antagonist (XT199)inhibited the pro-angiogenesis effect of either thyroid hormone analogsor T4-agarose. Data also demonstrated that the thyroid hormoneantagonist Tetrac inhibits the thyroid analog's pro-angiogenesisresponse. Thus, those thyroid hormone analogs tested are pro-angiogenic,an action that is initiated at the plasma membrane and involves αVβ3integrin receptors, and is MAPK-dependent.

The present invention describes a pro-angiogenesis promoting effect ofT₃, T₄, or T₄-agarose at sub-threshold levels of VEGF and FGF2 in an invitro three-dimensional micro-vascular endothelial sprouting model. Theinvention also provides evidence that the hormone effect is initiated atthe endothelial cell plasma membrane and is mediated by activation ofthe αVβ3 integrin and ERK1/2 signal transduction pathway.

Enhancement by T₃, T₄, or T₄ agarose of the angiogenesis activity of lowconcentrations of VEGF and FGF2 in the three-dimensional sprouting assaywas demonstrated. Either T₃, T₄ at 10⁻⁷-10⁻M, or T₄-agarose at 10⁻⁷ Mtotal hormone concentration was comparable in pro-angiogenesis activityto the maximal concentrations of VEGF and FGF2 effect in this in vitromodel. Although new blood vessel growth in the rat heart has beenreported to occur concomitantly with induction of myocardial hypertrophyby a high dose of T₄, thyroid hormone has not been regarded as anangiogenic factor. The present example establishes that the hormone inphysiologic concentrations is pro-angiogenic in a setting other than theheart.

T₄-agarose reproduced the effects of T₄, and this derivative of thyroidhormone is thought not to gain entry to the cell interior; it has beenused in our laboratory to examine models of hormone action for possiblecell surface-initiated actions of iodothyronines. Further, experimentscarried out with T₄ and tetrac also supported the conclusion that theaction of T₄ in this model was initiated at the plasma membrane. Tetracblocks membrane-initiated effects of T₄.

Since thyroid hormone non-genomically activates the MAPK (ERK1/2) signaltransduction pathway, the action of the hormone on angiogenesis can beMAPK-mediated. When added to the CAM model, an inhibitor of the MAPKcascade, PD 98059, inhibited the pro-angiogenic action of T₄. While thisresult was consistent with an action on transduction of the thyroidhormone signal upstream of an effect of T₄ on FGF2 elaboration, it isknown that FGF2 also acts via an MAPK-dependent mechanism. T₄ and FGF2individually cause phosphorylation and nuclear translocation of ERK1/2in endothelial cells and, when used in sub-maximal doses, combine toenhance ERK1/2 activation further. To examine the possibility that theonly MAPK-dependent component of hormonal stimulation of angiogenesisrelated exclusively to the action of FGF2 on vessel growth, cellularrelease of FGF2 in response to T₄ in the presence of PD 98059 wasmeasured. The latter agent blocked the hormone-induced increase ingrowth factor concentration and indicated that MAPK activation wasinvolved in the action of T₄ on FGF2 release from endothelial cells, aswell as the consequent effect of FGF2 on angiogenesis.

Effect of Thyroid Hormone on Angiogenesis

Either T₄, T₃, or T4-agarose at 0.01-0.1 μM resulted in significant(P<0.01) stimulation of angiogenesis (Table 4). This is shown to becomparable to the pro-angiogenesis efficacy of FGF2 (50 ng/ml) plus VEGF(25 ng/ml).

TABLE 4 In Vitro Pro-angiogenesis Effect of Growth Factors, ThyroidHormone, and Analogs in the Three-Dimensional Human Micro-vascularEndothelial Sprouting Assay Treatment Groups Mean Tube Vessel Length(mm) ± SD Control 0.76 ± 0.08  FGF2 (25 ng) + VEGF (50 ng) 2.34 ± 0.25*T3 (20 ng) 1.88 ± 0.21* T4 (23 ng) 1.65 ± 0.15* T4-agarose (23 ng) 1.78± 0.20* Data (means ± SD) were obtained from 3 experiments. Cells werepre-treated with Sub-threshold level of FGF2 (1.25 ng/ml) + VEGF(2.5ng/ml). Data represent mean ± SD, n = 3, *P < 0.01 by ANOVA, comparingtreated to control.

Effects of Tetrac on Thyroid Pro-Angiogenesis Action:

T₃ stimulates cellular signal transduction pathways initiated at theplasma membrane. These pro-angiogenesis actions are blocked by adeaminated iodothyronine analogue, tetrac, which is known to inhibitbinding of T₄ to plasma membranes. The addition of tetrac (0.1 μM)inhibited the pro-angiogenesis action of either T₃, T₄, or T₄-agarose(Tables 5-7). This is shown by the inhibition of number ofmicro-vascular endothelial cell migration and vessel length (Table 5-7).

Role of the ERK1/2 Signal Transduction Pathway in Stimulation ofAngiogenesis by Thyroid Hormone:

Parallel studies of ERK1/2 inhibition were carried out in thethree-dimensional micro-vascular sprouting assays. Thyroid hormone andanalog at 0.01-0.1 μM caused significant increase in tube length andnumber of migrating cells, an effect that was significantly (P<0.01)blocked by PD 98059 (Tables 5-7). This is shown by the inhibition ofnumber micro-vascular endothelial cell migration and vessel length(Table 5-7).

Role of the Integrin αVβ3 in Stimulation of Angiogenesis by ThyroidHormone:

Either T₃, T₄, or T₄-agarose at 0.01-0.1 μm-mediated pro-angiogenesis inthe presence of sub-threshold levels of VEGF and FGF2 was significantly(P<0.01) blocked by the αVβ3 integrin antagonist XT199 (Tables 5-7).This is shown by the inhibition of number of micro-vascular endothelialcell migration and vessel length (Table 5-7).

Thus, the pro-angiogenesis effect of thyroid hormone and its analogsbegins at the plasma membrane αVβ3 integrin and involves activation ofthe ERK1/2.

TABLE 5 Pro-angiogenesis Mechanisms of the Thyroid Hormone T₃ in theThree-Dimensional Human Micro-vascular Endothelial Sprouting Assay Meannumber of Migrated Mean vessel Length HDMEC treatment cells ± SD (mm) ±SD Control  88 ± 14 0.47 ± 0.06 T₃ (0.1 uM)  188 ± 15*  0.91 ± 0.04* T₃(0.1 uM) + PD98059 (3 ug) 124 ± 29 0.48 ± 0.09 T₃ (0.1 uM) + XT199 (2ug) 118 ± 18 0.47 ± 0.04 T₃ (0.1 uM) + tetrac (0.15 ug) 104 ± 15 0.58 ±0.07 Human dermal micro-vascular endothelial cells (HDMVC) were used.Cells were pretreated with FGF2 (1.25 ng/ml) + VEGF (2.5 ng/ml). Imageswere taken at 4 and 10X, day 3. Data represent mean ± SD, n = 3, *P <0.01.

TABLE 6 Pro-angiogenesis Mechanisms of the Thyroid Hormone T₄ in theThree-Dimensional Human Micro-vascular Endothelial Sprouting Assay Meannumber of Migrated Mean Vessel HDMEC treatment cells ± SD Length (mm) ±SD Control 88 ± 14 0.47 ± 0.06 T₄ (0.1 uM) 182 ± 11*  1.16 ± 0.21* T₄(0.1 uM) + PD98059 (3 ug) 110 ± 21  0.53 ± 0.13 T₄ (0.1 uM) + XT199 (2ug) 102 ± 13  0.53 ± 0.05 T₄ (0.1 uM) + Tetrac (0.15 ug) 85 ± 28 0.47 ±0.11 Human dermal micro-vascular endothelial cells (HDMVC) were used.Cells were pretreated with FGF2 (1.25 ng/ml) + VEGF (2.5 ng/ml). Imageswere taken at 4 and 10X, day 3. Data represent mean ± SD, n = 3, *P <0.01.

TABLE 7 Pro-angiogenesis Mechanisms of the Thyroid Hormone T₄-Agarose inthe Three-Dimension Human Micro-vascular Endothelial Sprouting AssayMean number of Migrated Mean Vessel Length HDMEC treatment cells ± SD(mm) ± SD Control 88 ± 14 0.47 ± 0.06 T₄-agarose (0.1 uM) 191 ± 13* 0.97 ± 0.08* T₄-agarose (0.1 uM) + PD98059 111 ± 8  0.56 ± 0.03 (3 ug)T₄-agarose (0.1 uM) + XT199 106 ± 5  0.54 ± 0.03 (2 ug) T₄-agarose (0.1uM) + Tetrac 87 ± 14 0.45 ± 0.09 (0.15 ug) Human dermal micro-vascularendothelial cells (HDMVC) were used. Cells were pretreated with FGF2(1.25 ng/ml) + VEGF (2.5 ng/ml). Images were taken at 4 and 10X, day 3.Data represent mean ± SD, n = 3, *P < 0.01.

Example 14 In Vitro Model for Evaluating Polymeric Thyroid AnalogsTransport Across the Blood-Brain Barrier

Described below is an in vitro method for evaluating the facility withwhich selected polymeric thyroid analog alone or in combination withnerve growth factor or other neurogenesis factors likely will passacross the blood-brain barrier. A detailed description of the model andprotocol are provided by Audus, et al., Ann. N.Y. Acad. Sci. 507: 9-18(1987), the disclosure of which is incorporated herein by reference.

Briefly, microvessel endothelial cells are isolated from the cerebralgray matter of fresh bovine brains. Brains are obtained from a localslaughter house and transported to the laboratory in ice cold minimumessential medium (“MEM”) with antibiotics. Under sterile conditions thelarge surface blood vessels and meninges are removed using standarddissection procedures. The cortical gray matter is removed byaspiration, then minced into cubes of about 1 mm. The minced gray matterthen is incubated with 0.5% dispase (BMB, Indianapolis, Ind.) for 3hours at 37° C. in a shaking water bath. Following the 3 hour digestion,the mixture is concentrated by centrifugation (1000×g for 10 min.), thenresuspended in 13% dextran and centrifuged for 10 min. at 5800×g.Supernatant fat, cell debris and myelin are discarded and the crudemicrovessel pellet resuspended in 1 mg/ml collagenase/dispase andincubated in a shaking water bath for 5 hours at 37° C. After the 5-hourdigestion, the microvessel suspension is applied to a pre-established50% Percoll gradient and centrifuged for 10 min at 1000×g. The bandcontaining purified endothelial cells (second band from the top of thegradient) is removed and washed two times with culture medium (e.g., 50%MEM/50% F-12 nutrient mix). The cells are frozen (−80° C.) in mediumcontaining 20% DMSO and 10% horse serum for later use.

After isolation, approximately 5×10⁵ cells/cm² are plated on culturedishes or 5-12 mm pore size polycarbonate filters that are coated withrat collagen and fibronectin. 10-12 days after seeding the cells, cellmonolayers are inspected for confluency by microscopy.

Characterization of the morphological, histochemical and biochemicalproperties of these cells has shown that these cells possess many of thesalient features of the blood-brain barrier. These features include:tight intercellular junctions, lack of membrane fenestrations, lowlevels of pinocytotic activity, and the presence of gamma-glutamyltranspeptidase, alkaline phosphatase, and Factor VIII antigenactivities.

The cultured cells can be used in a wide variety of experiments where amodel for polarized binding or transport is required. By plating thecells in multi-well plates, receptor and non-receptor binding of bothlarge and small molecules can be conducted. In order to conducttransendothelial cell flux measurements, the cells are grown on porouspolycarbonate membrane filters (e.g., from Nucleopore, Pleasanton,Calif.). Large pore size filters (5-12 mm) are used to avoid thepossibility of the filter becoming the rate-limiting barrier tomolecular flux. The use of these large-pore filters does not permit cellgrowth under the filter and allows visual inspection of the cellmonolayer.

Once the cells reach confluency, they are placed in a side-by-sidediffusion cell apparatus (e.g., from Crown Glass, Sommerville, N.J.).For flux measurements, the donor chamber of the diffusion cell is pulsedwith a test substance, then at various times following the pulse, analiquot is removed from the receiver chamber for analysis. Radioactiveor fluorescently-labelled substances permit reliable quantitation ofmolecular flux. Monolayer integrity is simultaneously measured by theaddition of a non-transportable test substance such as sucrose orinsulin and replicates of at least 4 determinations are measured inorder to ensure statistical significance.

Example 15 Traumatic Injury Model

The fluid percussion brain injury model was used to assess the abilityof polymeric thyroid hormone analogs alone or in combination with nervegrowth factors or other neurogenesis factors to restore central nervoussystem functions following significant traumatic brain injury.

I. Fluid Percussion Brain Injuries Procedure

The animals used in this study were male Sprague-Dawley rats weighing250-300 grams (Charles River). The basic surgical preparation for thefluid-percussion brain injury has been previously described. Dietrich,et al., Acta Neuropathol. 87: 250-258 (1994) incorporated by referenceherein. Briefly, rats were anesthetized with 3% halothane, 30% oxygen,and a balance of nitrous oxide. Tracheal intubation was performed andrats were placed in a stereotaxic frame. A 4.8-mm craniotomy was thenmade overlying the right parietal cortex, 3.8 mm posterior to bregma and2.5 mm lateral to the midline. An injury tube was placed over theexposed dura and bonded by adhesive. Dental acrylic was then pouredaround the injury tube and the injury tube was then plugged with agelfoam sponge. The scalp was sutured closed and the animal returned toits home case and allowed to recover overnight.

On the next day, fluid-percussion brain injury was produced essentiallyas described by Dixon, et al., J. Neurosurg. 67: 110-119 (1987) andClifton, et al., J. Cereb. Blood Flow Metab. 11: 114-121 (1991). Thefluid percussion device consisted of a saline-filled Plexiglas cylinderthat is fitted with a transducer housing and injury screw adapted forthe rat's skull. The metal screw was firmly connected to the plasticinjury tube of the intubated anesthetized rat (70% nitrous oxide, 1.5%halothane, and 30% oxygen), and the injury was induced by the descent ofa pendulum that strikes the piston. Rats underwent mild-to-moderate headinjury, ranging from 1.6 to 1.9 atm. Brain temperature was indirectlymonitored with a thermistor probe inserted into the right temporalismuscle and maintained at 37-37.5° C. Rectal temperature was alsomeasured and maintained at 37° C. prior to and throughout the monitoringperiod.

Behavioral Testing:

Three standard functional/behavioral tests were used to assesssensorimotor and reflex function after brain injury. The tests have beenfully described in the literature, including Bederson, et al., (1986)Stroke 17: 472-476; DeRyck, et al., (1992) Brain Res. 573: 44-60;Markgraf, et al., (1992) Brain Res. 575: 238-246; and Alexis, et al.,(1995) Stroke 26: 2338-2346.

A. The Forelimb Placing Test

Forelimb placing to three separate stimuli (visual, tactile, andproprioceptive) was measured to assess sensorimotor integration. DeRyck,et al., Brain Res. 573:44-60 (1992). For the visual placing subtest, theanimal is held upright by the researcher and brought close to a tabletop. Normal placing of the limb on the table is scored as “0,” delayedplacing (<2 sec) is scored as “1,” and no or very delayed placing (>2sec) is scored as “2.” Separate scores are obtained first as the animalis brought forward and then again as the animal is brought sideways tothe table (maximum score per limb=4; in each case higher numbers denotegreater deficits). For the tactile placing subtest, the animal is heldso that it cannot see or touch the table top with its whiskers. Thedorsal forepaw is touched lightly to the table top as the animal isfirst brought forward and then brought sideways to the table. Placingeach time is scored as above (maximum score per limb=4). For theproprioceptive placing subtest, the animal is brought forward only andgreater pressure is applied to the dorsal forepaw; placing is scored asabove (maximum score per limb=2). Finally, the ability of animals toplace the forelimb in response to whisker stimulation by the tabletopwas tested (maximum score per limb=2). Then subscores were added to givethe total forelimb placing score per limb (range=0-12).

B. The Beam Balance Test

Beam balance is sensitive to motor cortical insults. This task was usedto assess gross vestibulomotor function by requiring a rat to balancesteadily on a narrow beam. Feeney, et al., Science, 217: 855-857 (1982);Goldstein, et al., Behav. Neurosci. 104: 318-325 (1990). The testinvolved three 60-second training trials 24 hours before surgery toacquire baseline data. The apparatus consisted of a ¾-inch-wide beam, 10inches in length, suspended 1 ft. above a table top. The rat waspositioned on the beam and had to maintain steady posture with all limbson top of the beam for 60 seconds. The animals' performance was ratedwith the scale of Clifton, et al., J. Cereb Blood Flow Metab. 11:1114-121 (1991), which ranges from 1 to 6, with a score of 1 beingnormal and a score of 6 indicating that the animal was unable to supportitself on the beam.

C. The Beam Walking Test

This was a test of sensorimotor integration specifically examininghindlimb function. The testing apparatus and rating procedures wereadapted from Feeney, et al., Science, 217: 855-857 (1982). A 1-inch-widebeam, 4 ft. in length, was suspended 3 ft. above the floor in a dimlylit room. At the far end of the beam was a darkened goal box with anarrow entryway. At equal distances along the beam, four 3-inch metalscrews were positioned, angling away from the beam's center. A whitenoise generator and bright light source at the start of the beammotivated the animal to traverse the beam and enter the goal box. Onceinside the goal box, the stimuli were terminated. The rat's latency toreach the goal box (in seconds) and hindlimb performance as it traversedthe beam (based on a 1 to 7 rating scale) were recorded. A score of 7indicates normal beam walking with less than 2 foot slips, and a scoreof 1 indicates that the rat was unable to traverse the beam in less than80 seconds. Each rat was trained for three days before surgery toacquire the task and to achieve normal performance (a score of 7) onthree consecutive trials. Three baseline trials were collected 24 hoursbefore surgery, and three testing trials were recorded daily thereafter.Mean values of latency and score for each day were computed.

Example 16 Thyroid Hormone Analogs

Example 17 Retinoic Acid Analogs

Example 18 Thyroid Hormone Analog Conjugated with Retinoic Acid

Example 19 Halogenated Stilbestrol Analogs

Example 20 Compositions of T4 Analogs, Halogenated Stilbesterols, andRetinoic Acid

Example 21 Preparations of Compounds for Pet-Imaging

In general, the radioactive imaging agents of the present invention(Examples 16-20) are prepared by reacting radioactive 4-halobenzylderivatives with piperazine derivatives. Preferred are F-18 labeled4-fluorobenzyl derivatives for PET-imaging. A general method for thepreparation of 4-fluoro-.sup.18 F-benzyl halides is described in Iwataet al., Applied Radiation and Isotopes (2000), Vol. 52, pp. 87-92.

Example 22 Preparation of Compounds for SPECT-Imaging

For Single Photon Emission Computed Tomography (“SPECT”),^(99m)Tc-labeled compounds are preferred. A general synthetic pathwayfor these compounds starts with non-radioactive analogues of compoundsaccording to Examples 16-20 that are reacted with ^(99m)Tc-bindingchelators, e.g. N₂S₂-Chelators. The synthesis of the chelators followsstandard procedures, for example, the procedures described in A. Mahmoodet al., A N₂S₂-Tetradentate Chelate for Solid-Phase Synthesis:Technetium, Rhenium in Chemistry and Nuclear Medicine (1999), Vol. 5, p.71, or in Z. P. Zhuang et al., Bioconjugate Chemistry (1999), Vol. 10,p. 159.

One of the chelators is either bound directly to the nitrogen in the—N(R⁴)R⁵ group of the non-radioactive compounds according to Examples16-20, or via a linker moiety comprising an alkyl radical having one toten carbon atoms, wherein the alkyl radical optionally contains one toten —C(O)— groups, one to ten —C(O)N(R)— groups, one to ten —N(R)C(O)—groups, one to ten —N(R)— groups, one to ten —N(R)₂ groups, one to tenhydroxy groups, one to ten —C(O)OR— groups, one to ten oxygen atoms, oneto ten sulfur atoms, one to ten nitrogen atoms, one to ten halogenatoms, one to ten aryl groups, and one to ten saturated or unsaturatedheterocyclic rings wherein R is hydrogen or alkyl. A preferred linkermoiety is —C(O)—CH₂—N(H)—.

Example 23 T4 is a ligand of αVβ3 Integrin

To determine if T4 is a ligand of the αVβ3 integrin, 2 μg ofcommercially available purified protein was incubated with [¹²⁵I]T4, andthe mixture was run out on a non-denaturing polyacrylamide gel. αVβ3binds radiolabeled T4 and this interaction was competitively disruptedby unlabeled T4, which was added to αVβ3 prior to the [¹²⁵I]T4incubation, in a concentration-dependent manner (FIG. 24). Addition ofunlabeled T4 reduced binding of integrin to the radiolabeled ligand by13% at a total T4 concentration of 10⁻⁷ M total (3×10⁻¹⁰ M free T4), 58%at 10⁻⁶ M total (1.6×10⁻⁹ M free), and inhibition of binding was maximalwith 10⁻⁵ M unlabeled T4. Using non-linear regression, the interactionof αVβ3 with free T4 was determined to have a Kd of 333 pM and an EC₅₀of 371 pM. Unlabeled T3 was less effective in displacing[¹²⁵I]T4-binding to αVβ3, reducing the signal by 28% at 10⁻⁴ M total T3.

Example 24 T4 Binding to αVβ3 is Blocked by tetrac, RGD Peptide andIntegrin Antibody

We have shown previously that T4-stimulated signaling pathways activatedat the cell surface can be inhibited by the iodothyronine analog tetrac,which is shown to prevent binding of T4 to the plasma membrane. In ourradioligand-binding assay, while 10⁻⁸ M tetrac had no effect on[¹²⁵I]T4-binding to purified αVβ3, the association of T4 and αVβ3 wasreduced by 38% in the presence of 10⁻⁷ M tetrac and by 90% with 10⁻⁵ Mtetrac (FIG. 25). To determine specificity of the interaction, an RGDpeptide, which binds to the extracellular matrix-biding site on αVβ3,and an RGE peptide, which has a glutamic acid residue instead of anaspartic acid residue and thus does not bind αVβ3, were added in anattempt to displace T4 from binding with the integrin. Application of anRGD peptide, but not an RGE peptide, reduced the interaction of [¹²⁵I]T4with αVβ3 in a dose-dependent manner (FIG. 25).

To further characterize the interaction of T4 with αVβ3, antibodies toαVβ3 or αVβ5 were added to purified αVβ3 prior to addition of [¹²⁵I]T4.Addition of 1 μg/ml of αVβ3 monoclonal antibody LM609 reduced complexformation between the integrin and T4 by 52%, compared to untreatedcontrol samples. Increasing the amount of LM609 to 2 μg, 4 μg, and 8μg/ml diminished band intensity by 64%, 63% and 81%, respectively (FIG.26). Similar results were observed when a different αVβ3 monoclonalantibody, SC7312, was incubated with the integrin. SC7312 reduced theability of T4 to bind αVβ3 by 20% with 1 μg/ml of antibody present, 46%with 2 μg, 47% with 4 μg, and by 59% when 8 μg/ml of antibody werepresent. Incubation with monoclonal antibodies to αV and β3, separately,did not affect [¹²⁵I]T4-binding to αVβ3, suggesting that the associationrequires the binding pocket generated from the heterodimeric complex ofαVβ3 and not necessarily a specific region on either monomer. To verifythat the reduction in band intensity was due to specific recognition ofαVβ3 by antibodies, purified αVβ3 was incubated with a monoclonalantibody to αVβ5 (P1F6) or mouse IgG prior to addition of [¹²⁵I]T4,neither of which influenced complex formation between the integrin andradioligand (FIG. 26).

Example 25 T4-Stimulated MAPK Activation is Blocked by Inhibitors ofHormone Binding and of Integrin αVβ3

Nuclear translocation of phosphorylated MAPK (pERK1/2) was studied inCV-1 cells treated with physiological levels of T4 10⁻⁷ M total hormoneconcentration, 10⁻¹⁰ M free hormone) for 30 min. Consistent with resultswe have previously reported, T4 induced nuclear accumulation ofphosphorylated MAPK in CV-1 cells within 30 min (FIG. 27).Pre-incubation of CV-1 cells with the indicated concentrations of αVβ3antagonists for 16 h reduced the ability of T4 to induce MAPK activationand translocation. Application of an RGD peptide at 10⁻⁸ and 10⁻⁷ M hada minimal effect on MAPK activation. However, 10⁻⁶ M RGD peptideinhibited MAPK phosphorylation by 62% compared to control cultures andactivation was reduced maximally when 10⁻⁵ M RGD (85% reduction) and10⁻⁴ M RGD (87% reduction) were present in the culture media. Additionof the nonspecific RGE peptide to the culture media had no effect onMAPK phosphorylation and nuclear translocation following T4 treatment inCV-1 cells.

Tetrac, which prevents the binding of T4 to the plasma membrane, is aneffective inhibitor of T4-induced MAPK activation. When present at aconcentration of 10⁻⁶ M with T4, tetrac reduced MAPK phosphorylation andtranslocation by 86% when compared to cultures treated with T4 alone(FIG. 27). The inhibition increased to 97% when 10⁻⁴ M tetrac was addedto the culture media for 16 h before the application of T4. Addition ofαVβ3 monoclonal antibody LM609 to the culture media 16 h prior tostimulation with T4 also reduced T4-induced MAPK activation. LM609 at0.01 and 0.001 μg/ml of culture media did not affect MAPK activationfollowing T4 treatment. Increasing the concentration of antibody in theculture media to 0.1, 1, and 10 μg/ml reduced levels of phosphorylatedMAPK found in the nuclear fractions of the cells by 29%, 80%, and 88%,respectively, when compared to cells treated with T4 alone.

CV-1 cells were transiently transfected with siRNA to αV, β3 or both αVand β3 and allowed to recover for 16 h before being placed in serum-freemedia. Following T4 treatment for 30 min, the cells were harvested andeither nuclear protein or RNA was extracted. FIG. 28A demonstrates thespecificity of each siRNA for the target integrin subunit. CV-1 cellstransfected with either the αV siRNA or both αV and β3 siRNAs showeddecreased αV subunit RT-PCR products, but there was no difference in αVmRNA expression when cells were transfected with the siRNA specific forβ3, or when exposed to the transfection reagent in the absence ofexogenous siRNA. Similarly, cells transfected with β3 siRNA had reducedlevels of β3 mRNA, but relatively unchanged levels of αV siRNA. Theaddition of T4 for 30 min did not alter mRNA levels for either αV or β3,regardless of the siRNA transfected into the cells.

Activated MAPK levels were measured by western blot in CV-I cellstransfected with siRNAs to αV and β3, either individually or incombination (FIG. 28B). CV-I cells treated with scrambled negativecontrol siRNA had slightly elevated levels of T4-induced activated MAPKwhen compared to the parental cell line. Cells exposed to thetransfection reagent alone display similar levels and patterns of MAPKphosphorylation as the non-transfected CV-1 cells. When either αV siRNAor β3 siRNA, alone or in combination, was transfected into CV-1 cells,the level of phosphorylated MAPK in vehicle-treated cultures waselevated, but the ability of T4 to induce a further elevation inactivated MAPK levels was inhibited.

Example 26 Hormone-Induced Angiogenesis is Blocked by Antibody to αVβ3

Angiogenesis is stimulated in the CAM assay by application ofphysiological concentrations of T4 (FIG. 29A and summarized in FIG.29B). 10⁻⁷ M T4 placed on the CAM filter disk induced blood vesselbranch formation by 2.3-fold (P<0.001) when compared to PBS-treatedmembranes. Propylthiouracil, which prevents the conversion of T4 to T3,has no effect on angiogenesis caused by T4. The addition of a monoclonalantibody, LM609 (10 μg/filter disk), directed against αVβ3, inhibitedthe pro-angiogenic response to T4.

Example 27 Biocompatible Polymer Conjugates of Thyroid Hormone Analogsfor Short and Long-Term Delivery Sketch 1: Thyroid Hormone Analogs:

Sketch 2: Molecular Models Showing the 3 D view for Topography &Molecular Geometry in stick, ball & stick, disc and space filling modelsfor comparative evaluation. Stick Model: Molecules in two sets—showingmolecular density in lower set.

Ball & Stick Model: Triac and Tetrac—lower set showing molecular densityand topographical alignment.

Disc Model: Triac and Tetrac

Space Filling Model: Triac and Tetrac

Thyroid Hormone Analog and Anti-Cancer Activity:

The thyroid hormone metabolites Triac and Tetrac are used in treatmentof thyroid cancer to enrich and as substitute therapy for its needs.

Commercially Available Thyroid Hormones & its Analogs:

There are several brand names synthetic thyroid hormones available inthe market including Unithroid®, Levothroid®, Synthroid® and Levoxyl®.There are generic formulations like Levo-T®, Levothyroxine Sodium® andNovothyrox®.

Natural Thyroid hormones are sold as food supplement in a dried andpowdered form obtained from slaughtered animal's thyroid glands. Thisdesiccated product pill may contain unwanted animal protein, improperbalance of the T3 and T4 compounds and synthetic binders which is leastrecommended for human consumption. The ratio of T3 to T4 may vary foreach batch depending on the animal's gland and has not been found to beconstant for any proper delivery in human subjects. The complication indose, delivery and availability in the system argues for a standardized,constant, limited dose and temporal release regimen for safe humanconsumption and other topical uses.

Thyroid Hormone Regulated Release System:

As of today, no specific and standardized regimen of treatment forthyroid hormone replacement therapy is available to patients in UnitedStates. We are proposing a long term, permanent slow release system ofindividual thyroid constituents based on personalized needs presortedfor the dose, desired activity and response profile in test systems. Wewill be achieving it through the polymer bound thyroid constituentsdelivery to the site of these products for dose defining and in alimited distribution. The proposed conjugates (FIG. 3—a sketch) willhave both short and longer term release capacity achieved throughhydrolysable and non-hydrolysable characteristics. This will achieve theminimum dose level delivery for the specific cardio vascular and woundhealing properties.

Sketch 3: Ordered & Random Polymer Conjugates—

Conjugated Delivery Systems:

Among the developing delivery systems, synthetic polymers conjugatedchemical entities are gaining ground. A variety of synthetic, naturaland biopolymeric origin side groups with efficient biodegradablebackbone polymers are available and are in use in the trade. Poly alkylglycols, polyesters, poly anhydride, poly saccharide, and poly aminoacids are available for conjugation depending on the finalcharacteristic of the conjugated product in terms of theirhydrophilicity, hydrophobic nature, hydrolyses by enzymes, co-factorsand biologically available acids in vivo as well as in vitro systems forthe purpose.

Polymer Conjugation-Synthesis & Purification:

We are proposing a library of conjugated products (see FIG. 6 andTable-1) for the controlled, hydrolysable as well as non-hydrolysablepolymer conjugate products including the biopolymers. A test case willevolve from each category of polymer products for their detailed studyin terms of its pharmacokinetics covering delivery, transport, half-lifeand degradation and or erosion data. The series will also be tried toprepare as a concurrent or library design of combinatorial synthesisfitting into the similar chemical class of reaction for the substrateand varying polymers utilizing DCC, DCC/HOBt based and other watersoluble reagents including placement of linker and commonly used NHSester based synthetic strategies with a view for future developments inthe polymeric conjugation combinatorial or parallel synthesis. Theavailable facilities of the synthesizer at PRI will be utilized towardsthis effect and each product will be individually purified forsuitability of the biological test systems.

Polymer Conjugates—Physical & Chemical Characterization:

A through spectro-analytical and chromatographic analysis of the polymerbound compounds will be performed using NMR (High & Low Field—Proton &Carbon, DEPT, HOMOCOSY & HETEROCOSY/HETCOR wherever applicable), IR, MS,HPLC, thermal and environmental degradation analysis for stabilitysuitability and degradation rate profile.

Release & Stability Studies:

A detailed HPLC analyses for quantitative release analyses will beundertaken based on our established protocol for the individual thyroidproducts i.e., GC-1, T3, T4 and DITPA as well as Triac and Tetrac alongwith individual polymer and polymer conjugated product's chromatographicand spectro-analytical profile.

Polymers Compatibility Criteria for Conjugation:

Biodegradable and biocompatible polymers have been designated asprobable carriers for long term and short time delivery vehiclesincluding non hydrolysable polymeric conjugates (table 1). PEGs and PEOsare the most common hydroxyl end polymers with a wide range of molecularweights to choose for the purpose of solubility (easy carrier mode),degradation times and ease of conjugation. One end protectedMethoxy-PEGs will also be employed as a straight chain carrier capableof swelling and thereby reducing the chances of getting protein attachedor stuck during the subcellular transportation. Certain copolymers ofethylene and vinyl acetate, i.e. EVAc which have exceptionally goodbiocompatibility, low crystallinity and hydrophobic in nature are idealcandidate for encapsulation mediated drug delivery carrier.

Polymers with demonstrated high half-life and in-system retentionproperties will be undertaken for conjugation purpose. Among the mostcommon and recommended biodegradable polymers from lactic and glycolicacids will be used. The copolymers of L-lactide, and L-lysine is usefulbecause of its availability of amine functional groups for amide bondformation and this serves as a longer lasting covalent bonding site ofthe carrier and transportable thyroid compound linked together throughthe carboxyl moiety in all the thyroid constituents.

The naturally occurring polysaccharides from cellulose, chitin, dextran,ficoll, pectin, carrageenan (all subtypes), and alginate and some oftheir semi-synthetic derivatives are ideal carriers due to its highbiocompatibility, bio systems familiar degradation products (monosaccharide from glucose and fructose), hydrophilic nature, solubility,protein immobilization/interaction for longer term stability of thepolymer matrix. This provides a shell for extra protection for polymermatrix from degradation over time and adding to the effective half lifeof the conjugate.

Protein & Polypeptide from serum albumin, collagen, gelatin andpoly-L-lysine, poly-L-alanine, poly-L-serine are natural amino acidsbased drug carrier with advantage of biodegradation, biocompatibilityand moderate release times of the carrier molecule. Poly-L-serine is offurther interest due to its different chain derivatives, e.g., polyserine ester, poly serine imine and conventional poly serine polymericbackbone with available sites for specific covalent conjugation.

Synthetic hydrogels from methacrylate derived polymers have beenfrequently used in biomedical applications because of their similarityto the living tissues. The most widely used synthetic hydrogels arepolymers of acrylic acid, acrylamide and 2-hydroxyethyl methacrylate(HEMA). The poly HEMA are inexpensive, biocompatible, available primaryalcohol side chain elongation functionality for conjugation and fit forocular, intraocular and other ophthalmic applications which makes themperfect drug delivery materials. The pHEMA are immune to cell attachmentand provides zero cell motility which makes them an ideal candidate forinternal delivery system.

Synthetic thyroid analog DITPA conjugation library design program hasbeen achieved with the development of crude DITPA conjugated products.PVA and PEG hydrophilic polymer coupling mediated through DicycolhexylCarbodiimide and by other coupling reagents of hydrophilic andhydrophobic nature is under progress.

The design for the evolution of library synthesis on solid phasesynthesizer is in its final stages and a model for its high throughputscreening (HTS) will be put in place based on commonality of testingsystem and parametric criteria. The statistical analyses for thedelivery time, half-life and conceived stability of the conjugates willbe accumulated for the purpose of structure delivery analyses (SDA).Following is a list of intended polymer conjugates for preparation(Table 8).

TABLE 8 Library of Designated Polymer Conjugates for PossiblePreparation based on Chemical Class Reactivities & Stability Data.Properties (H Hydrolysable, Sr. NH Non Hydrolysable, No. Polymer RRRetarded Release) 1 PEO H 2 m-PEG H 3 PVA Hydrophilic, H 4 PLLAHydrophilic, H 5 PGA Hydrophilic, H 6 Poly L-Lysine NH 7 Human SerumAlbumin Protein, NH 8 Cellulose Derivative Polysaccharide, RR(Carbomethoxy/ethyl/ hydroxypropyl) 9 Hyaluronic Acid Polysaccharide, RR10 Folate Linked Cyclodextrin/Dextran RR 11 Sarcosine/Amino Acid spacedRR Polymer 12 Alginate/Carrageenan Polysaccharide, RR 13 Pectin/ChitosanPolysaccharide, RR 14 Dextran Polysaccharide, RR 15 Collagen Protein, NH16 Poly amine Aminic, NH 17 Poly aniline Aminic, NH 18 Poly alaninePeptidic, RR 19 Polytryptophan Peptidic, NH/RR 20 Polytyrosine Peptidic,NH/RR

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

1. A method of treating a condition amenable to treatment by promotingangiogenesis, said method comprising administering to a subject in needthereof an amount of a thyroid hormone, thyroid hormone analog, orpolymeric forms thereof, effective for promoting angiogenesis in saidsubject.
 2. The method of claim 1, wherein said condition amenable totreatment by promoting angiogenesis is selected from the groupconsisting of: occlusive vascular disease, coronary disease, erectiledysfunction, myocardial infarction, ischemia, stroke, peripheral arteryvascular disorders, and wounds.
 3. The method of claim 1, wherein thethyroid hormone or analog is conjugated to a member selected from thegroup consisting of: polyvinyl alcohol, acrylic acid ethyleneco-polymer, polyethyleneglycol (PEG), polylactic acid, and agarose. 4.The method of claim 1, wherein the thyroid hormone analog islevothyroxine (T4), triiodothyronine (T3),3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl)-phenoxy acetic acid(GC-1), or 3,5-diiodothyropropionic acid (DITPA).
 5. The method of claim1, wherein the mode of administration of the thyroid hormone, thyroidhormone analog, or polymeric forms thereof, is parenteral, oral, rectal,topical, or combinations thereof.
 6. The method of claim 5, wherein saidparenteral administration is subcutaneous, intraperitoneal,intramuscular, intravenous, or combinations thereof.
 7. The method ofclaim 1, wherein the thyroid hormone, thyroid hormone analog, orpolymeric forms thereof is encapsulated or incorporated in amicroparticle, liposome, or polymer.
 8. The method of claim 7, whereinthe polymer is polyglycolide, polylactide, or co-polymers thereof. 9.The method of claim 7, wherein the liposome or microparticle has a sizeof less than about 200 nm.
 10. The method of claim 7, wherein theliposome or microparticle is administered intravenously.
 11. The methodof claim 10, wherein the liposome or microparticle is lodged incapillary beds surrounding ischemic tissue.
 12. The method of claim 1,wherein the thyroid hormone, thyroid hormone analog, or polymeric formsthereof is administered via catheter.
 13. The method of claim 12,wherein the thyroid hormone, thyroid hormone analog, or polymeric formsthereof is present in a polymeric system applied to the inside of ablood vessel via said catheter.
 14. The method of claim 1, wherein thethyroid hormone, thyroid hormone analog, or polymeric forms thereof isco-administered with one or more compounds selected from the groupconsisting of: a growth factor, a vasodilator, an anti-coagulant, andcombinations thereof.
 15. The method of claim 14, wherein said growthfactor is selected from the group consisting of: transforming growthfactor alpha (TGFα), transforming growth factor beta (TGFβ), basicfibroblast growth factor, vascular endothelial growth factor, epithelialgrowth factor, nerve growth factor, platelet-derived growth factor, andvascular permeability factor.
 16. The method of claim 14, wherein saidvasodilator is adenosine, adenosine derivatives, or combinationsthereof.
 17. The method of claim 14, wherein said anticoagulant isheparin, heparin derivatives, anti-factor Xa, anti-thrombin, aspirin,clopidgrel, or combinations thereof.
 18. The method of claim 14, whereinthe thyroid hormone, thyroid hormone analog, or polymeric forms thereofis administered as a bolus injection prior to or post-administering saidgrowth factor, vasodilator, anti-coagulant, or combinations thereof. 19.A method for promoting angiogenesis along or around a medical device,said method comprising coating the device with a thyroid hormone,thyroid hormone analog, or polymeric forms thereof, prior to insertingthe device into a patient.
 20. The method of claim 19, wherein saidcoating step further comprises coating the device with a growth factor,a vasodilator, an anti-coagulant, or combinations thereof.
 21. Themethod of claim 19, wherein said medical device is a stent, a catheter,a cannula, or an electrode.
 22. A method for treating a conditionamenable to treatment by inhibiting angiogenesis, said method comprisingadministering to a subject in need thereof an amount of ananti-angiogenesis agent effective for inhibiting angiogenesis in saidsubject.
 23. The method of claim 22, wherein said condition amenable totreatment by inhibiting angiogenesis is selected from the groupconsisting of: a primary or metastatic tumor, glioma, breast cancer, anddiabetic retinopathy.
 24. The method of claim 22, wherein saidanti-angiogenesis agent is tetraiodothyroacetic acid (TETRAC),triiodothyroacetic acid (TRIAC), monoclonal antibody LM609, XT 199 orcombinations thereof.
 25. The method of claim 22, wherein the mode ofadministration of the anti-angiogenesis agent is parenteral, oral,rectal, topical, or combinations thereof.
 26. The method of claim 22,wherein the anti-angiogenesis agent is co-administered with one or moreother anti-angiogenesis therapies or chemotherapeutic agents.
 27. Themethod of claim 22, wherein the anti-angiogenesis agent acts at the cellsurface.
 28. An angiogenic agent comprising a thyroid hormone or analogthereof.
 29. An angiogenic agent comprising a thyroid hormone or analogthereof conjugated to a polymer.
 30. The angiogenic agent of claim 29,wherein said analog is selected from those compounds recited in FIG. 20,Tables A-D.
 31. The angiogenic agent of claim 29, wherein said thyroidhormone analog is levothyroxine (T4), triiodothyronine (T3),3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl)-phenoxy acetic acid(GC-1), or 3,5-diiodothyropropionic acid (DITPA).
 32. The angiogenicagent of claim 29, wherein said polymer is polyvinyl alcohol, acrylicacid ethylene co-polymer, polyethyleneglycol (PEG), polylactic acid, oragarose.
 33. The angiogenic agent of claim 29, wherein said conjugationis via a covalent or non-covalent bond.
 34. The angiogenic agent ofclaim 33, wherein said covalent bond is an ester linkage or an anhydridelinkage.
 35. A pharmaceutical formulation comprising the angiogenicagent of claim 29 in a pharmaceutically acceptable carrier.
 36. Thepharmaceutical formulation of claim 35, further comprising one or morepharmaceutically acceptable excipients.
 37. The pharmaceuticalformulation of claim 35, wherein said agent is encapsulated orincorporated in a microparticle, liposome, or polymer.
 38. Thepharmaceutical formulation of claim 37, wherein the liposome ormicroparticle has a size less than 200 nm.
 39. The pharmaceuticalformulation of claim 35, wherein said formulation has a parenteral,oral, rectal, or topical mode of administration, or combinationsthereof.
 40. The pharmaceutical formulation of claim 35, wherein saidformulation is co-administered to a subject in need thereof with one ormore compounds selected from the group consisting of: a growth factor, avasodilator, an anti-coagulant, and combinations thereof.
 41. Apharmaceutical composition for treating neurodegenerative diseases ordisorders comprising a therapeutically effective amount of one or morethyroid hormone analogs.
 42. The pharmaceutical composition of claim 41,wherein said thyroid hormone analog is T3, DITPA, or GC-1.
 43. Thepharmaceutical composition of claim 41, wherein said thyroid hormoneanalog is polymer conjugated.
 44. The pharmaceutical composition ofclaim 43, wherein said polymer is selected from the group consisting of:polyvinyl alcohol, polylactic acid, acrylic anhydride, polyethyleneglycol, cellulose, and dendrimers.
 45. The pharmaceutical composition ofclaim 41, wherein said thyroid hormone analog is conjugated tonanoparticles.
 46. The pharmaceutical composition of claim 41, whereinsaid composition further comprises pro-angiogenesis factors, nervegrowth factors, neurogenesis factors, anti-inflammatory agents,antioxidants, or combinations thereof.
 47. The pharmaceuticalcomposition of claim 46, wherein said pro-angiogenesis factor is FGF orVEGF.
 48. The pharmaceutical composition of claim 46, wherein saidantioxidant is vitamin C, vitamin E, resveratrol-like compounds, orcombinations thereof.
 49. The pharmaceutical composition of claim 46,wherein said anti-inflammatory agent is a compound selected from thegroup consisting of non-steroidal compounds, insulin sensitizers, andprotesome inhibitors.
 50. The pharmaceutical composition of claim 41,wherein the composition further comprises a pharmaceutically acceptableexcipient.
 51. The pharmaceutical composition of claim 41, wherein saidtherapeutically effective amount is a dosage of between 1 to 100 mg. 52.The pharmaceutical composition of claim 41, wherein saidneurodegenerative disease or disorder is selected from the groupconsisting of: motor neuron defects, amyotrophic lateral sclerosis,multiple sclerosis, spinal cord injury, demyelinating diseases,myelopathies.
 53. A method of treating a neurodegenerative disease ordisorder, said method comprising administering to a subject in needthereof, a pharmaceutically effective amount of a pharmaceuticalcomposition comprising one or more thyroid hormone analogs.
 54. Themethod of claim 53, wherein the pharmaceutical composition isco-administered with nerve growth factors, neurogenesis factors,antioxidants, anti-inflammatory agents, or combinations thereof.
 55. Amethod of repairing a damaged neural pathway of the peripheral nervoussystem or of the central nervous system, said method comprisingadministering to a subject in need thereof, a pharmaceutically effectiveamount of a pharmaceutical composition comprising one or more thyroidhormone analogs.
 56. An imaging agent for diagnosing a neurodegenerativedisease comprising a labeled thyroid hormone analog that binds totransthyretin.
 57. The imaging agent of claim 56, wherein said agentpasses the blood brain barrier.
 58. The imaging agent of claim 56,wherein said thyroid hormone analog is selected from the groupconsisting of: T3, T4 DITPA, GC-1.
 59. The imaging agent of claim 56,wherein said thyroid hormone analog is polymer conjugated.
 60. Theimaging agent of claim 56, wherein said thyroid hormone analog isconjugated to a dendrimer.
 61. The imaging agent of claim 56, whereinsaid neurodegenerative disease is Alzheimer's disease.
 62. The imagingagent of claim 61, wherein said agent inhibits the formation of amyloidfibrils.
 63. The imaging agent of claim 56, wherein said agent is imagedby positron emission tomography, single photon emission computedtomography, or magnetic resonance imaging.
 64. The imaging agent ofclaim 56, wherein said thyroid hormone analog is conjugated to retinoicacid, halogenated stilbestrols, or analogs thereof.
 65. A method fordiagnosing Alzheimer's disease, the method comprising, administering toa subject suspected of having, or at risk for, Alzheimer's disease animaging agent comprising a labeled thyroid hormone analog that binds totransthyretin, and forming at least one image showing the distributionof the imaging agent within the brain of the subject using a brainimaging technique selected from the group consisting of: positronemission tomography, single photon emission computed tomography, ormagnetic resonance imaging